Nuclear factors associates with transcriptional regulation

ABSTRACT

Constitutive and tissue-specific protein factors which bind to transcriptional regulatory elements of Ig genes (promoter and enhancer) are described. The factors were identified and isolated by an improved assay for protein-DNA binding. Genes encoding factors which positively regulate transcription can be isolated and employed to enhance transription of Ig genes. In particular, NF-kB, the gene encoding NF-kB, IkB and the gene encoding IkB and uses therefor.

GOVERNMENT SUPPORT

The work leading to this invention was supported in part by a grant fromthe National Cancer Institute. The Government has certain rights in thisinvention.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 07/791,898filed Nov. 13, 1991, now abandoned, which is a continuation-in-part ofSer. No. 06/946,365 (WHI86-10), filed Dec. 24, 1986, now abandoned; Ser.No. 07/318,901 (WHI87-11A), filed Mar. 3, 1989, now abandoned; Ser. No.07/162,680 (WHI87-11), filed Mar. 1, 1988, now abandoned; and Ser. No.07/341,436 (WHI89-02), filed Apr. 21, 1989, now abandoned; Ser. No.06/817,441 (MIT-4167), filed Jan. 9, 1986, now abandoned; Ser. No.07/155,207 (MIT-4167A), filed Feb. 12, 1988, now abandoned; and Ser. No.07/280,173 (MIT-4167AA), filed Dec. 5, 1988, now abandoned. All of theabove applications are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

Trans-acting factors that mediate B cell specific transcription ofimmunoglobulin (Ig) genes have been postulated based on an analysis ofthe expression of exogenously introduced Ig gene recombinants inlymphoid and non-lymphoid cells. Two B cell-specific, cis-actingtranscriptional regulatory elements have been identified. One element islocated in the intron between the variable and constant regions of bothheavy and kappa light chain genes and acts as a transcriptionalenhancer. The second element is found upstream of both heavy chain andkappa light chain gene promoters. This element directs lymphoid-specifictranscription even in the presence of viral enhancers.

Mouse and human light chain promoters contain the octamer sequenceATTTGCAT approximately 70 base pairs upstream from the site ofinitiation. Heavy chain gene promoters contain the identical sequence ininverted orientation, ATGCAAAT, at the same position. This elementappears to be required for the efficient utilization of Ig promoters inB cells. The high degree of sequence and positional conservation of thiselement as well as its apparent functional requirement suggests itsinteraction with a sequence-specific transcription factor but no suchfactor has been identified.

DISCLOSURE OF THE INVENTION

This invention pertains to human lymphoid-cell nuclear factors whichbind to gene elements associated with regulation of the transcription ofIg genes and to methods for identification and for isolation of suchfactors. The factors are involved in the regulation of transcription ofIg genes. The invention also pertains to the nucleic acid encoding theregulatory factors, to methods of cloning factor-encoding genes and tomethods of altering transcription of Ig genes in lymphoid cells orlymphoid derived cells, such as hybridoma cells, by transfecting orinfecting cells with nucleic acid encoding the factors.

Four different factors which bind to transciptional regulatory DNAelements of Ig genes were identified and isolated in nuclear extracts oflymphoid cells. Two of the factors, IgNF-A and E, are constitutive; twoIgNF-B and κ-3 (hereinafter NF-κB) are lymphoid cell specific. Eachfactor is described below.

IgNF-A (NF-A1)

IgNF-A binds to DNA sequences in the upstream regions of both the murineheavy and kappa light chain gene promoters and also to the murine heavychain gene enhancer. The binding is sequence specific and is probablymediated by a highly conserved sequence motif, ATTTGCAT, present in allthree transcriptional elements. A factor with binding specificitysimilar to IgNF-A is also present in human HeLa cells indicating thatIgNF-A may not be tissue specific.

E factors

The E factors are expressed in all cell types and bind to the light andheavy chain enhancers.

IgNF-B (NF-A2)

IgNF-B exhibits the same sequence-specificity as IgNF-A; it binds toupstream regions of murine heavy and kappa light chain gene promotersand to murine heavy chain gene enhancer. This factor, however, islymphoid specific; it is restricted to B and T cells.

NF-κB (Previously Kappa-3)

NF-κB binds exclusively to the kappa light chain gene enhancer (thesequence TGGGGATTCCCA). Initial work provided evidence that NF-kB isspecific to B-lymphocytes (B-cells) and also to be B-cell stagespecific. NF-kB was originally detected because it stimulatestranscription of genes encoding kappa immunoglobulins in B lymphocytes.As described herein, it has subsequently been shown that transcriptionfactor NF-kB, previously thought to be limited in its cellulardistribution, is, in fact, present and inducible in many, if not all,cell types and that it acts as an intracellular messenger capable ofplaying a broad role in gene regulation as a mediator of induciblesignal transduction. It has now been demonstrated that NF-kB has acentral role in regulation of intercellular signals in many cell types.For example, NF-kB has now been shown to positively regulate the humanβ-interferon (β-IFN) gene in many, if not all, cell types. As describedbelow, it is now clear not only that NF-kB is not tissue specific innature, but also that in the wide number of types of cells in which itis present, it serves the important function of acting as anintracellular transducer of external influences. NF-kB has been shown tointeract with a virus inducible element, called PRDII, in the β-IFN geneand to be highly induced by virus infection or treatment of cells withdouble-stranded RNA. In addition, NF-kB controls expression of the humanimmunodeficiency virus (HIV).

As further described, it has been shown that a precursor of NF-KB ispresent in a variety of cells, that the NF-KB precursor in cytosolicfractions is inhibited in its DNA binding activity and that inhibitioncan be removed by appropriate stimulation, which also results intranslocation of NF-KB to the nucleus. A protein inhibitor of NF-KB,designated IkB, has been shown to be present in the cytosol and toconvert NF-KB into an inactive form in a reversible, saturable andspecific reaction. Release of active NF-kB from the IkB-NF-kB complexhas been shown to result from stimulation of cells by a variety ofagents, such as bacterial lipopolysaccharide, extracellular polypeptidesand chemical agents, such as phorbol esters, which stimulateintracellular phosphokinases. IkB and NF-KB appear to be present in astoichiometric complex and dissociation of the two complex componentsresults in two events: activation (appearance of NF-KB binding activity)and translocation of NF-KB to the nucleus.

Identification and Isolation of the Transcriptional Regulatory Factors

The transcription regulatory factors of the present invention wereidentified and isolated by means of a modified DNA binding assay. Theassay has general applicability for analysis of protein DNA interactionsin eukaryotic cells. In performing the assay, DNA probes embodying therelevant DNA elements or segments thereof are incubated with cellularnuclear extracts. The incubation is performed under conditions whichallow the formation of protein-DNA complexes. Protein-DNA complexes areresolved from uncomplexed DNA by electrophoresis through polyacrylamidegels in low ionic strength buffers. In order to minimize binding ofprotein in a sequence nonspecific fashion, a competitor DNA species canbe added to the incubation mixture of the extract and DNA probe. In thepresent work with eukaryotic cells the addition of alternating copolymerduplex poly(dI-dC)-poly(dI-dC) as a competitor DNA species provided foran enhancement of sensitivity in the detection of specific protein-DNAcomplexes and facilitated detection of the regulatory factors describedherein.

This invention pertains to the transcriptional regulatory factors, thegenes encoding the four factors associated with transcriptionalregulation, reagents (e.g., oligonucleotide probes, antibodies) whichinclude or are reactive with the genes or the encoded factors and usesfor the genes, factors and reagents. It further relates to NF-KBinhibitors, including isolated IkB, the gene encoding IkB and agents ordrugs which enhance or block the activity of NF-KB or of the NF-KBinhibitor (e.g., IkB).

The invention also pertains to a method of cloning DNA encoding thetranscriptional regulatory factors or other related transcriptionalregulatory factors. The method involves screening for expression of thepart of the binding protein with binding-site DNA probes. Identificationand cloning of the genes can also be accomplished by conventionaltechniques. For example, the desired factor can be purified from crudecellular nuclear extracts. A portion of the protein can then besequenced and with the sequence information, oligonucleotide probes canbe constructed and used to identify the gene coding the factor in a cDNAlibrary. Alternatively, the polymerase chain reaction (PCR) can be usedto identify genes encoding transcriptional regulatory factors.

The present invention further relates to a method of inducing expressionof a gene in a cell. In the method, a gene of interest (i.e., one to beexpressed) is linked to the enhancer sequence containing the NF-KBbinding site in such a manner that expression of the gene of interest isunder the influence of the enhancer sequence. The resulting constructincludes the kappa enhancer or a kappa enhancer portion containing atleast the NF-KB binding site, the gene of interest, and a promoterappropriate for the gene of interest. Cells are transfected with theconstruct and, at an appropriate time, exposed to an appropriate inducerof NF-KB, resulting in induction of NF-KB and expression of the gene ofinterest.

The subject invention further relates to methods of regulating (inducingor preventing) activation of NF-KB, controlling expression of theimmunoglobulin kappa light chain gene and of other genes whoseexpression is controlled by NF-KB (e.g., HIV).

As a result of this finding, it is now possible to alter or modify theactivity of NF-κB as an intracellular messenger and, as a result, toalter or modify the effect of a variety of external influences, referredto as inducing substances, whose messages are transduced within cellsthrough NF-κB activity. Alteration or modification, whether to enhanceor reduce NF-κB activity or to change its binding activity (e.g.,affinity, specificity), is referred to herein as regulation of NF-κBactivity. The present invention relates to a method of regulating orinfluencing transduction, by NF-κB, of extracellular signals intospecific patterns of gene expression and, thus, of regulatingNF-κB-mediated gene expression in the cells and systems in which itoccurs.

In particular, the present invention relates to a method of regulating(enhancing or diminishing) the activity of NF-κB in cells in which it ispresent and capable of acting as an intracellular messenger, as well asto substances or composition useful in such a method. Such methods andcompositions are designed to make use of the role of NF-κB as a mediatorin the expression of genes in a variety of cell types. The expression ofa gene having a NF-κB binding recognition sequence can be regulated,either positively or negatively, to provide for increased or decreasedproduction of the protein whose expression is mediated by NF-κB.NF-κB-mediated gene expression can also be selectively regulated byaltering the binding domain of NF-κB in such a manner that bindingspecificity and/or affinity are modified. In addition, genes which donot normally possess NF-κB binding recognition sequences can be placedunder the control of NF-κB by inserting an NF-κB binding site in anappropriate position, to produce a construct which is then regulated byNF-κB. As a result of the present invention, cellular interactionsbetween NF-κB and a gene or genes whose expression is mediated by NF-κBactivity and which have, for example, medical implications (e.g.,NF-κB/cytokine interactions; NF-κB/HTLV-I tax gene product interactions)can be altered or modified.

Genes encoding the regulatory factors can be used to alter cellulartranscription. For example, positive acting lymphoid specific factorsinvolved in Ig gene transcription can be inserted into Ig-producingcells in multiple copies to enhance Ig production. Genes encoding tissuespecific factors can be used in conjunction with genes encodingconstitutive factors, where such combinations are determined necessaryor desirable. Modified genes, created by, for example, mutagenesistechniques, may also be used. Further, the sequence-specific DNA bindingdomain of the factors can be used to direct a hybrid or altered proteinto the specific binding site.

DNA sequences complementary to regions of the factor-encoding genes canbe used as DNA probes to determine the presence of DNA encoding thefactors for diagnostic purposes and to help identify other genesencoding transcriptional regulatory factors. Antibodies can be raisedagainst the factors and used as probes for factor expression. Inaddition, the cloned genes permit development of assays to screen foragonists or antagonists of gene expression and/or of the factorsthemselves. Further, because the binding site for NF-kB in the kappagene is clearly defined, an assay for blockers or inhibitors of bindingis available, as is an assay to determinte whether active NF-κB ispresent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of the 5'0 region of the MOPC 41 V.sub.κgene segment;

FIG. 2 shows the nucleotide sequences of actual and putative bindingsites of IgNF-A;

FIG. 3 shows the DNA sequence of the promoter region of MODC41;

FIG. 4 shows a restriction map of the μ-enhancer;

FIG. 5A shows a restriction map of the μ300 fragment;

FIG. 5B is a restriction map of the relevant region;

FIG 6 provides a summary of these results.

FIG. 7 is a restriction map of κ enhancer;

FIG. 8 shows the binding analysis of NK-κB in cells at various stages ofB-cell differentiation.

FIG. 9 shows the λgt11-EBNA-1 (λEB) recombinant and the oriP probe.

FIG. 10 shows the sequence of the DNA probe used to screen for an H2TF1and NF-κB expression.

FIG. 11A shows the nucleotide sequence of the oct-2 gene derived fromcDNA and the predicted amino acid sequence of encoded proteins.

FIG. 11B shows the nucleotide sequence of the 3' terminus and predictedthe amino acid sequence of the C-terminus derived from clone pass-3.

FIGS. 11C-11E are a schematic representation of the amino acid sequencededuced from oct-2 gene derived cDNA.

FIG. 12 is a schematic representation of expression plasmidpBS-ATG-oct-2.

FIG. 13 shows amino acid sequence alignment of the DNA binding domain ofoct-2 factor with homeo-boxes of several other genes.

FIG. 14 is a representation of binding sites for the NF-kB transcriptionfactor in the immunoglobulin kappa light chain enhancer and the HIVenhancer. Boxes indicate the binding sites for NF-kB (B); otherregulatory sites are referred to as E1l E2 and E3 and Spl. Dots indicateguanosine residues in the kappa enhancer whose methylation interferedwith binding of NF-kB.

FIGS. 15A and 15B show results which characterize the NF-kB protein.FIG. 15A represents determination of the molecular weight of NF-kB.Nuclear extract (300 ug of protein) from TPA-stimulated 70Z/3 pre-Bcells was denatured and subjected to reducing SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). Protein in the molecular weight fractionsindicated by dashed lines was eluted and renatured prior to mobilityshift assays as described. A fluorogram of a native gel is shown. Thefilled arrow-head indicates the position of a specific proteinDNA-complex only detected in the 62-55 kDa fraction with a wild type(wt) but not with a mutant (mu) kappa enhancer fragment. The openarrowhead indicates the position of unbound DNA-fragments. FIG. 15B is arepresentation of glycerol gradient centrifugation of NF-kB. Nuclearextract (400 ug of protein) from TPA-stimulated 70Z/3 cells wassubjected to ultracentrifugation on a continuous 10-30% glycerolgradient for 20 hours at 150,000×g in buffer D(+). Co-sedimentedmolecular weight standards (ovalbumin, 45 kDa; bovine serum albumin, 67kDa; immunoglobulin G, 158 kDa; thyroglobulin monomer, 330 kDa and dimer660 kDa) were detected in the fractions by SDS-PAGE, followed byCoomassie Blue staining. The distribution of NF-kB activity wasdetermined by electrophoretic mobility shift assays using anend-labelled kappa enhancer fragment. Fluorograms of native gels areshown. The specificity of binding was tested using a kappa enhancerfragment with a mutation in the NF-kB binding site.

FIG. 16 shows a titration and kinetic analysis of the in vitroinactivation of NF-kB. NF-kB contained in nuclear extracts fromTPA-treated 70Z/3 cells (2.2 μg of protein) was incubated withincreasing amounts (0.25 to 2.25 μg of protein) of a gel filtrationfraction containing IkB. After the DNA binding reaction, samples wereanalyzed by EMSA. The ³² P-radioactivity in the NF-kB-DNA complexesvisualized by fluorography was determined by liquid scintillationcounting. All reactions were performed in triplicates. The barsrepresent standard deviations.

FIG. 17 is a diagram showing the location of positive regulatory domainII (PRDII) within the interferon gene regulatory element (IRE) and acomparison of the nucleotide sequences of the PRDII site, κB site, andthe H2TF1 site.

FIGS. 18A and 18B demonstrate the functional interchangeability of PRDIIand NF-κB in vivo.

FIG. 18A is an autoradiogram showing the results of CAT assays ofextracts prepared from L929 and S194 myeloma cells transfected with thereporter genes illustrated in FIG. 18B.

FIG. 18B is a diagram of the reporter genes containing multiple copiesof PRDII or κB. Two or four PRDII sites (P)₂ and (P)₄, respectively!were inserted upstream of the truncated -41 human β-globin promoter/CATfusion gene (-41β) using an oligonucleotide containing two copies ofPRDII (PRDIIx2, as described in the Exemplification). Two copies of asynthetic wild-type κB site, or mutant κB site (B and B⁻, respectively)were inserted upstream of the mouse c-fos promoter/CAT fusion gene inwhich the promoter was truncated to nucleotide -56 (Δ56).

CLONE DEPOSITS

Clones λh3 and λ3-1 were deposited (Feb. 12, 1988) at the AMerican TypeCulture Collection (12301 Parklawn Drive; Rockville, Md. 20852), underthe terms of the Budapest Treaty. They were assigned ATCC Designationals67629 and 67630, respectively. Upon issue of a U.S. patent from thesubject application, all restrictions upon the availability of theseclones will be irrevocably removed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the identification, isolation andcharacterization of human transcriptional regulatory factors, to genesencoding the factors, methods of isolating DNA encoding transcriptionalregulatory factors and the encoded factors, uses of the DNA, encodedfactors, and antibodies against the encoded factors, inhibitors of thetranscriptional regulatory factors. In particular, it relates to thetranscriptional regulatory factor NF-kB (previously designated Kappa-3);its inhibitor; IkB, DNA encoding each, methods of altering interactionsof NF-kB and IkB. and methods of regulating the activity of NF-kB. Asdescribed herein, NF-kB, was initially thought to be a B-cell specificfactor involved in immunoglobulin gene regulation and has since beenshown to be inducible in many, if not all, cell types and to act as anintracellular transducer or mediator of a variety of externalinfluences. The following is a description of the discovery andcharacterization of four transcriptional regulatory factors, assessmentof the function of NF-kB and its role, in many cell types, as anintracellular mediator or transducer of a variety of externalinfluences, discovery of the NF-kB inhibitor IkB and demonstration thatNF-kB and IkB exist in the cytoplasm as a NF-kB-IkB complex whosedissociation results in activation of NF-kB and its translocation intothe nucleus. The following is also a description of the uses of thegenes, regulatory factors and related products and reagents.

The transcriptional regulatory factors described herein can be broadlyclassified as constitutive (non-lymphoid) or tissue (lymphoid) specific.All factors are believed to play a role in transcription ofimmunoglobulin (Ig) genes. Constitutive factors, which are present innon-lymphoid cells, may have a role in regulating transcription of genesother than Ig genes; lymphoid-specific factors might also play a role inregulating transcription of genes in addition to Ig genes.

Four transcriptional regulatory factors were identified, as describedbelow and in the Examples. The presence of constitutive factors renderedthe detection of tissue specific factors more difficult. A sensitive DNAbinding assay, described below, was employed in all studies tofacilitate detection of tissue specific factors.

The characteristics of the transcriptional regulatory factors IgNF-A, E,IgNFB and Kappa-3 (or NF-κB) are summarized in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        CHARACTERISTICS OF FOUR                                                       TRANSCRIPTIONAL REGULATORY FACTORS                                                   Ig Regulatory Sequence                                                 Factor   Promoter  Enhancer                                                   Designation                                                                            V.sub.H                                                                              V.sub.κ                                                                        U.sub.E                                                                            K.sub.E                                                                            Lymphoid                                                                             Nonlymphoid                           ______________________________________                                        IgNF-A   +      +      +    -    +      +                                     (NF-A1)                                                                       E factors                                                                              -      -      +    +    +      +                                     IgNF-B   +      +      +    -    +      -                                     (NF-A2)                                                                       Kappa-3  -      -      -    +    +      -                                     (NF-κB)                                                                 ______________________________________                                    

Factor Ig NF-A

As indicated in Table 1, IgNF-A binds to Ig regulatory DNA elements inthe region of mouse heavy and kappa light chain gene promoters and alsoto mouse heavy chain gene enhancer. DNAase I footprint analysisindicates that the binding is mediated by the octamer sequence(ATTTGCAT) which occurs in mouse and human light chain gene promotersapproximately 70 base pairs upstream from the site of initation and inheavy chain gene promoters at about the same position (in invertedsequence).

Deletion or disruption of the IgNF-A binding site in Ig promoterssignificantly reduces the level of accurately initiated transcripts invivo. See, e.g., Bergman, Y. et al. PNAS U.S.A. 81 7041-7045 (1984);Mason, J. O. et al. Cell 41 479-487 (1985). As demonstrated in Example2, this also occurs in an in vitro transcription system. IgNF-A appearsto be a positive transacting factor.

The IgNF-A binding site appears to be a functional component of theB-cell-specific Ig promoter. For example, sequences from this promotercontaining the IgNF-A binding site specify accurate transcription inB-cells but not in Hela cells. IgNF-A however, may not be restricted toB-cells because a factor was detected in Hela cell extracts whichgenerated complexes with similar mobilities and sequence specificity (astested by competition analysis). Interestingly, the Ig octamer motif inthe IgNF-A binding site has recently been shown to be present in theupstream region (about 225 bp) of vertebrate U1 and U2 snRNA genes. Moreimportantly, this element dramatically stimulates (20 to 50 fold)transcription of U2 snRNA genes in Xenopus oocytes. Therefore, IgNF-Amay be a constitutive activator protein that also functions in the highlevel expression of U1 and U2 snRNA genes in vertebrate cells.

The presence of an IgNF-A binding site in the mouse heavy chain enhancersuggests the additional involvement of IgNF-A in enhancer function. Itis known that deletion of an 80 bp region of the enhancer containing theputative binding site reduces activity approximately tenfold. Theoccupation of the binding site, in vivo, has been inferred from the factthat the G residue in the enhancer octamer is protected from dimethylsulfate modification only in cell of the B lineage. Furthermore, IgNF-Aalso binds in a sequence-specific manner to the SV40 enhancer (J.Weinberger, personal communication), which contains the Ig octamermotif, thereby strengthening the notion that the factor participates inenhancer function.

E Factors

The E factors are constitutive factors which bind to the Ig light andheavy chain enhancer.

Factor Ig NF-B

Factor IgNF-B binds to the same regulatory elements as IgNF-A. Indeed,the binding site for IgNF-B appears to be the octamer motif. In contrastto IgNF-A, IgNF-B is lymphoid cell specific. It was found in nuclearextracts from pre-B, mature B and myeloma cell lines and in nuclearextracts from some T cell lymphomas. IgNF-B was undetectable in nuclearextracts of several non-lymphoid cells. The gene encoding Ig NF-B hasbeen cloned (oct-2 clone below) and its nucleotide sequence has beendetermined (See FIG. 11A).

Factor NF-KB

NF-κB (previously referred to as Kappa-3) binds only to the Ig lightchain enhancer. The binding is mediated by the sequence TGGGATTCCCA. Thefactor initially was characterized as lymphoid cell specific and also aslymphoid stage specific; that is, work showed that it is expressed onlyby mature B-cells. Thus, it is a marker of B cell maturation (e.g. thefactor can be used to type B cell lymphomas). Additional work, describedin Examples 8-15 in particular, has shown that NF-kB is an induciblefactor in cells, both pre-B and non pre-B, in which it is notconstitutively present (Example 8), that it is present in the cytoplasmas an inactive precursor (Examples 10 and 11), and that the inactiveprecursor is a complex of NF-kB and an inhibitor, referred to as IkB,which converts NF-kB to an inactive form in a reversible saturable andspecific reaction. Dissociation of the complex results in activation ofNF-kB (appearance of NF-kB binding activity) and translocation of theNF-kB into the nucleus.

As discussed below, it is now evident that this DNA binding protein,initially thought to be a B-cell specific factor and subsequentlyimplicated in gene regulation in T lymphocytes, is present in many, ifnot all, cell types and that it acts as an important intracellulartransducer or mediator of a variety of external influences. That is,NF-κB is now known to be involved in a variety of induction processes inessentially all types of cells and is thought to participate in a systemthrough which multiple induction pathways work, in much the same manneras "second messengers" (e.g., cAMP, IP3) act, resulting in transductionof a variety of extracellular signals into specific patterns of geneexpression. Different cell types and different genes respond to this onesignal, which serves as a central "control", whose activity can bealtered by means of the present invention. As used, the terms alteringand modifying mean changing the activity or function of NF-κB in such amanner that it differs from the naturally-occurring activity of NF-κBunder the same conditions (e.g., is greater than or less than, includingno activity, the naturally-occurring NF-κB activity; is of differentspecificity in terms of binding, etc.).

It has been shown that NF-κB participates in gene expression (e.g.,cytokine gene expression) which is activated by a specific influence orextracellular signal (e.g., infection by a virus) in many, if not alltypes of cells. In particular, it has now been demonstrated that NF-κBhas a central role in virus induction of human β-interferon (β-IFN) geneexpression. Virus infection has been shown to potently activate thebinding and nuclear localization of NF-κB and, in pre-B lymphocytes, toresult in expression of both the β-IFN gene and the Ig kappa gene. Thewide variety of cell types in which β-interferon can be induced and thedivergent set of gene induction processes which involve NF-κB provideevidence that NF-κB plays a broad role in gene regulation as a mediatorof inducible signal transduction.

The following is a description and exemplification of work (Example 15)which clearly demonstrates the role of NF-κB in virus-induced humanβ-IFN gene expression; of the evidence that there is a single NF-κBwhich serves many roles in many different cell types and how it acts asan intracellular messenger in a variety of different gene inductionprocesses, particularly several which have important effects on cellphysiology in health and disease; and of the use of methods andcompositions of the present invention.

Role of NF-κB in Cytokine Gene Regulation

The role of NFκB as a mediator or messenger in cytokine gene regulationhas been demonstrated, as explained in greater detail in theExemplification, through assessment of the viral induction of humanβ-IFN gene expression. The human β-IFN gene has been shown to bepositively regulated by NF-κB, which was, in turn, shown to interactwith a virus inducible element, called PRDII, in the β-IFN gene. Asdescribed below, NF-κB has been shown to be highly induced in lymphoidand non-lymphoid cells by either virus infection or treatment of cellswith double-stranded RNA poly (rI:rC)!. It has also been shown to bindspecifically to PRDII, which is one of two positive regulatory domainsof the interferon gene regulatory element (IRE) which, together with therelease of a negative influence over a site called NRDI, are necessaryand sufficient for virus induction of the β-IFN gene.

It is known that the human β-interferon (β-IFN) gene is highly inducibleby virus or synthetic double-stranded RNA poly(rI:rC) in many, if notall, cell types. DeMaeyer, E. and J. DeMaeyer-Guignard, "Interferons andOther Regulatory Cytokines", John Wiley and Sons, New York (1988).Extensive characterization of the β-IFN gene promoter has revealed acomplex arrangement of positive and negative regulatory elements.Taniguchi, T., Ann. Rev. Immunol., 6:439-464 (1988). A 40 base pair DNAsequence designated the IRE (Interferon gene Regulatory Element) is bothnecessary and sufficient for virus induction. Goodbourn et al., Cell,41: 509-520 (1985). The IRE contains two distinct positive regulatorydomains (PRDI and PRDII) and one negative regulatory domain (NRDI).Goodbourn et al., Cell, 45:601-610 (1986); Goodbourn, S. and T.Maniatis, Proc. Natl. Acad. Sci. USA, 85:1447-1451 (1988). Virusinduction apparently requires cooperative interactions between PRDI andPRDII. Goodbourn, S. and T. Maniatis, Proc. Natl. Acad. Sci. USA,85:1447-1451 (1988). Single copies of PRDI or PRDII alone are notsufficient for virus or poly(rI:rC) induction, but two or more copies ofPRDI (Fujita et al., Cell , 49:357-367 (1987)) or PRDII (Fan, C. M. andT. Maniatis, EMBO J., 8:101-110 (1989)) confer inducibility onheterologous promoters.

The PRDII sequence binds a nuclear factor, designated PRDII-BF, that ispresent in extracts from both uninduced and induced MG63 cells. Keller,A. and T. Maniatis, Proc. Natl. Acad. Sci. USA, 85:3309-3313 (1988). AcDNA clone encoding a PRDII binding factor (designated PRDII-BF1) wasisolated. DNA sequence analysis revealed that PRDII-BF1 is similar, ifnot identical, to a cDNA clone encoding a protein that binds to relatedsites in both the MHC class I H-2K^(b) gene and the Ig κ enhancer. Singhet al., Cell 52:415-423 (1988). This observation suggested that PDRIImight be functionally related to the H2-K^(b) and κ enhancer sites.

The site in the H-2K^(b) promoter is required for its constitutive andinterferon-induced expression and binds a factor designated H2TF1 andpossibly similar factors KBF1 and EBP-1 which are constitutivelyexpressed in most cell types. (Baldwin, A. S. and P. A. Sharp, Proc.Natl. Acad. Sci. USA, 85:723-727 (1988); Yano et al., EMBO J.,6:3317-3324 (1988); Clark et al., Genes & Dev., 2:991-1002 (1988)). TheIg κ enhancer site, termed κB, binds NF-κB, which is required for κenhancer function. Sen, R. and D. Baltimore, Cell, 46:705-716 (1986);Atchison, M. and R. P. Perry, Cell, 48:121-128 (1987); Lenardo, M. etal., Science, 236:1573-1577 (1987). The transcriptional activities andin vitro binding of the κB site and PRDII were compared and resultsshowed that the two regulatory sequences are interchangeable in vivo,and that PRDII specifically binds NF-κB in vitro. A binding activityindistinguishable from NF-κB in nuclear extracts from virus-infectedcells was also identified. Viral treatment of 70Z/3 pre-B lymphocytesinduced κ gene expression as well as β-IFN gene expression. Theseresults show that NF-κB plays an important role in the virus inductionof the β-IFN gene and indicate that NF-κB acts similarly to secondmessenger systems in that it transduces a variety of extracellularsignals into specific patterns of gene expression.

It has been shown, by all available criteria, that the κB and the PRDIIDNA elements--one from the Ig κ light chain gene and one from the β-IFNgene--are interchangeable. They drive transcription of reporter genes inresponse to the same set of inducers, cross-compete for binding in vitroand have closely-related DNA sequences. Another indication of theidentity of the two elements is that release of NF-κB from a complexwith its inhibitor, I-κB, correlates with the induction of β-IFN in L929cells and that, conversely, a β-IFN inducer (Sendai virus) induces κgene transcription in 70Z/3 cells. This relationship is strengthened bythe correlation between the ability of mutations in PRDII to decreaseβ-IFN gene inducibility in vivo and reduce binding to NF-κB in vitro.Evidence that double-stranded RNA induces a factor resembling NF-κB hasalso been recently obtained by Visvanathan, K. V. and S. Goodbourn, EMBOJ., 8:1129-1138 (1989).

Results described in the Exemplification strongly imply that β-IFN geneexpression is activated, at least in part, by induction of NF-κB. Theability of NF-κB to be activated by a protein synthesis-independentpathway is consistent with the fact that induction of β-IFN is notblocked by cycloheximide. In fact, the β-IFN gene, like the κ gene, canbe induced by cycloheximide. Ringold et al., Proc. Natl. Acad. Sci. USA,81:3964-3968 (1984); Enoch et al., Mol. Cell Biol., 6:801-810 (1987);and Wall et al., Proc. Natl. Acad. Sci. USA, 83:295-298 (1986). Inaddition to the interaction between NF-κB and PRDII, virus induction ofβ-IFN involves activation through PRDI and the release of repression atNRDI. The present data revealing a role for NF-κB in β-IFN regulation isa striking example of how it is used in many, if not all, cell types.

Evidence for the Existence of a Single NF-κB

As shown in Table 1, sites present in a variety of genes form a mobilityshift electrophoretic complex which resembles NF-κB, as reported by Sen,and Baltimore, upon incubation of the Ig κ enhancer with B-cellextracts, Sen, R. and D. Baltimore, (Cell 46:705-716 (1986)). Thebiochemical evidence suggests the involvement of a single NF-κB in allcell types and not a family of factors in which individual members arespecifically inducible in particular cell types.

This evidence includes the fact that purification of NF-κB tohomogeneity from both human and bovine sources yields a singlepolypeptide chain of approximately 44 to 50 kD (although this could be afragment of a larger protein). Kawakami et al., Proc. Natl. Acad. Sci.USA 85:4700-4704 (1988); and Lenardo et al., Proc. Natl. Acad. Sci. USA,85:8825-8829 (1988). NF-κB adopts an oligomeric structure in solution;based on the size of the complex, it exists either as a homodimer orassociates with a heterologous subunit of approximately equal molecularweight. Baeuerle, P. et al., Cold Spring Harbor Symposium, 53:789-798(1988); Lenardo et al., Proc. Natl. Acad. Sci. USA, 85:8825-8829 (1988).NF-κB has the unique property that nucleoside triphosphates dramaticallystimulate its ability to bind DNA in vitro. Lenardo et al., Proc. Natl.Acad. Sci. USA, 85:8825-8829 (1988). NF-κB is further distinguished bythe fact that it can be released as an active binding species from aninactive cytosolic form that is completed with IκB. Baeuerle, P. and D.Baltimore, Cell, 53:211-217 (1988); Baeuerle, P. and D. Baltimore,Science, 242:540-545 (1988). All of these features are shared by theNF-κB complex irrespective of the cell-type from which it is derived.

More importantly, no differences in binding specificity have beendetected between the NF-κB complexes from different cell types. That is,the NF-κB complex induced in T cells has no preference for sites fromgenes activated in T cells rather than those from genes activated in Bcells and vice-versa. Lenardo et al., Proc. Natl. Acad. Sci. USA,85:8825-8829 (1988). An identical pattern of base contacts ischaracteristic of complexes between DNA and NF-κB from different celltypes, further decreasing the possibility that the NF-κB complex indifferent cell types is due to heterogeneous proteins.

It is clear that NF-κB binding sites are recognized by other obviouslydistinct transcription factors. The best examples are the H2-TF1 andKBF-1 proteins, which bind to an NF-κB-like site in the H2-K^(b) MHCclass I gene (Baldwin, A. S. and P. A. Sharp, Mol. Cell; Biol.,7:305-313 (1987); and Yano et al., EMBO J., 6:3317-3324 (1987)).However, these factors are constitutively active nuclear bindingproteins in many different cell types and no evidence implicates them ininducible gene expression. Other examples include the factor EBP-1 whichbinds to the SV40 κB site but has a different molecular size than NF-κBand is also not inducible (Clark et al., Genes & Development, 2:991-1002(1988); HIVEN86A, an 86 kD factor identified in activated T cellextracts by DNA affinity chromatography (Franza et al., Nature,330:391-395 (1987)); and finally, a protein encoded by a cDNA (λh3 orPRDII-BF1) selected from λgt11 expression libraries (Singh et al., Cell,52:415-523 (1988)). Recent evidence has made it unlikely that the λh3clone encodes NF-κB because several cell types that have abundantexpression of NF-κB lack the transcript for λh3. Taken together thesefindings indicate that there is only one NF-κB that serves multipleroles in many different cell types.

NF-κB Acts as an Intracellular Messenger

A salient feature of the induction of NF-κB is that it takes place inthe absence of new protein synthesis. Sen, R. and D. Baltimore, Cell,47:921-928 (1986). In fact, the protein synthesis inhibitorcycloheximide can alone activate NF-κB. Sen, R. and D. Baltimore, Cell,47:921-928 (1986). It appears, therefore, that NF-κB induction involvesthe conversion of a pre-existing precursor into an active form.

Inactive NF-κB is complexed with a labile inhibitor protein, I-κB.Cytosolic extracts from uninduced cells can be treated in vitro withdissociating agents such as formamide and deoxycholate to unmask veryhigh levels of NF-κB activity. Baeuerle, P. and D. Baltimore, Cell,53:211-217 (1988). These treatments by and large do not work on nuclearextracts from uninduced cells. Conversely, NF-κB activated normally inthe cell is detected in nuclear but not cytosolic extracts implying anuclear translocation step following activation in vivo. The inhibitoryactivity has been shown to be due to a protein of 68 kD that can beseparated chromatographically from NF-κB. Baeuerle, P. and D. Baltimore,Science, 242:540-545 (1988). This protein is able to inhibit the bindingof NF-κB but not other DNA-binding proteins and has therefore been named"I-κB" (Inhibitor-κB).

Notably, crude preparations of I-κB efficiently inhibit binding of NF-κBderived from mature B cells or other cell-types that have been induced.Baeuerle, P. and D. Baltimore, Science, 242:540-545 (1988). Theimplication is that activation of NF-κB involves a modification of I-κBand not NF-κB. This distinguishes NF-κB activation from a similarphenomenon involving the glucocorticoid receptor. In the latter, adirect interaction of glucocorticoid with the receptor is required torelease it from a cytoplasmic complex with the heat shock protein,hsp90. Picard, D. and K. R. Yamamoto, EMBO J., 6:3333-3340 (1987).

The model which ties together these observations is that NF-κB isinitially located in the cytoplasm in a form unable to bind DNA becauseit is complexed with I-κB. Various inducers then cause an alteration inI-κB allowing NF-κB to be released from the complex. Free NF-κB thentravels to the nucleus and interacts with its DNA recognition sites tofacilitate gene transcription. The complex formation of NF-κB with I-κBappears to be rapidly and efficiently reversible in vitro which lendsitself well to the shut-off as well as turn-on of NF-κB binding.Moreover, this model resolves a major question in signal transduction:NF-κB, like the glucocorticoid receptor, acts as a messenger to transmitthe gene induction signal from the plasma membrane to the nucleus.

The model presented above is not unlike the well known role of cAMP as asecond messenger in the action of many hormones; in the case of cAMP,the first messenger is the hormone itself. (see, eg., Pastan, Sci.Amer., 227:97-105 (1972)). The essential features of the cAMP model arethat cells contain receptors for hormones in the plasma membrane. Thecombination of a hormone with its specific membrane receptor stimulatesthe enzyme adenylate cyclase which is also bound to the plasma membrane.The concomitant increase in adenylate cyclase activity increases theamount of cAMP inside the cell which serves to alter the rate of one ormore cellular processes. An important feature of this second messengeror mediator model is that the hormone (the first messenger) need notenter the cell.

The participation of NF-κB in gene expression that is activated inspecific cells by specific influences calls for a level of regulation inaddition to the inducibility of NF-κB binding. How is "cross-talk"between the various paths employing NF-κB avoided? Factors acting uponother sequences within a transcriptional control element appear togovern the response to the NF-κB signal, as described herein for β-IFN.Studies of β-IFN expression have shown that virus induction worksthrough three events: two virus-inducible positive signals, one of whichis NF-κB, and the release of a single negative regulator. The twopositive signals work through distinct DNA sites (PRDI and PRDII), butmust act together to facilitate transcription. Either site alone is notinducible.

The theme of multiple signals that generate specificity is furthersupported by studies of the Ig κ gene and the IL-2 receptor gene. TheNF-κB site from the κ light chain enhancer alone on a shortoligonucleotide will stimulate transcription in B and T lymphocytes aswell as in non-lymphoid cells. Pierce, J. W. et al., Proc. Natl. Acad.Sci. USA, 85:1482-1486 (1988). Its function depends solely on thepresence of NF-κB. By contrast, the entire κ enhancer is inducible onlyin B lymphocytes and is unresponsive to NF-κB in other cell types. Therestricted response to NF-κB by the κ enhancer has now been attributedto a repressor sequence. The repressor sequence resides in the enhancersome distance away from the NF-κB binding site and acts to suppresstranscriptional effects of NF-κB in non-B cell types. The activation ofthe IL-2 receptor gene specifically in T lymphocytes is attained by aslightly different means. Full induction of this gene depends on NF-κBas well as a positively-acting sequence immediately downstream. Thedownstream element has now been found to bind a T cell specific proteincalled NF-ILT. Though NF-ILT is not itself inducible, its presence onlyin T cells seems to contribute to T cell specific induction of the IL-2receptor gene.

Role of NF-κB in Other Inducible Systems

Recently, NF-κB has been implicated in several other inducible systems.For example, NF-κB is induced in T-cells by a trans-activator (tax) ofHTLV-1 or by PMA/PHA treatment and thereby activates the IL-2 receptor αgene and possibly the IL-2 gene. Bohnlein et al., Cell, 53:827-836(1988); Leung, K. and G. Nabel, Nature, 333:776-778 (1988); Ruben etal., Science, 241:89-92 (1988); Cross et al., Science, (1989); andLenardo et al., Proc. Natl. Acad. Sci. USA, 85:8825-8829 (1988). NF-κBalso appears to take part in gene activation during the acute phaseresponse of the liver. Edbrooke et al., (1989). Results described heresuggest that inducibility of NF-κB plays a prominent role ininteractions between cytokines. IL-1 and TNF-α activate NF-κB bindingand both have been shown known to induce β-IFN. Osborn et al., Proc.Natl. Acad. Sci. USA, 86:2336-2340 (1989); and DeMaeyer, E. and J.DeMaeyer-Guignard, "Interferons and Other Regulatory Cytokines", JohnWiley and Sons, New York (1988). Finally, NF-κB has been shown to play arole in the transcription of human immunodeficiency virus (HIV). Nabel,G. and D. Baltimore, Nature, 326:711-713 (1987). Significantly, herpessimplex virus has recently been shown to increase HIV LTR transcriptionthrough NF-κB/core sequences. Gimble et al., J. Virol., 62:4104-4112(1988). Thus, NF-κB induction may lead to the propagation of HIV incells infected with other viruses.

NF-κB is unique among transcription regulatory proteins in its role as amajor intracellular transducer of a variety of external influences inmany cell types. In the cases studied thus far, it appears that theactual target of induction is I-κB, which becomes modified to a formthat no longer binds to NF-κB. Baeuerle, P. and D. Baltimore, Science,242:540-545 (1988). The released NF-κB then displays DNA bindingactivity and translocates to the nucleus.

Role of NF-kB in HIV Expression

Treatment of latently HIV-infected T-cells with phorbol ester(12-O-tetradecanoylphorbol 13-acetate; TPA) and with phytohaemaglutinin(PHA) results in the onset of virus production. Harada, S. et al.,Virology, 154:249-258 (1986); Zagury, D. J. et al., Science, 232:755-759(1986). The same treatments induce NF-kB activity in the humanT-lymphoma cell line Jurkat. Sen, R. and D. Baltimore, Cell, 47:921-928(1896). This correlation and the finding that two NF-kB binding sitesare present upstream of the transcriptional start site in the HIVenhancer, (FIG. 14) suggested a direct role for NF-kB in the activationof the viral enhancer, an event ultimately leading to the production ofvirus. Nabel, G. and D. Baltimore, Nature, 326:711-713 (1987). Thispossibility was tested by transient transfection of a plasmid containingan HIV LTR-controlled CAT gene into a human T-lymphoma cell line. Nabel,G. and D. Baltimore, Nature, 326:711-713 (1987). The viral cis-actingelements rendered the transcriptional activity of the CAT generesponsive to TPA/PHA treatment of cells. This inducible transcriptionalstimulation of the CAT gene was completely dependent on intact bindingsites for NF-kB in the HIV enhancer because mutation of the two bindingsites abolished inducibility. A protein-DNA complex with a fragment ofthe HIV enhancer containing the two NF-kB binding sites was observed inmobility shift assays only with nuclear extracts from TPA/PHA-stimulatedT-cells and not with control extracts. These observations providedstrong evidence that HIV expression in latently infected T-cells isinduced by the same transcription factor that regulates kappa geneexpression, NF-kB. A precursor of NF-kB is constitutively present inT-cells. Its activity can be induced by a treatment that mimicksantigenic T-cell activation and, after induction, NF-kB is able to bindto and subsequently enhance the activity of HIV transcriptional controlelements. Thus, it is reasonable to conclude that NF-kB is thephysiological transactivator responsible for initial expression ofdormant HIV-DNA following stimulation of T-lymphocytes.

Other factors have also been implicated in the control of HIV expressionincluding the HIV-encoded tat-III protein, the cellular transcriptionfactor Spl, and viral proteins encoded by the ElA gene of adenovirus andthe ICPO gene of the Herpes Simplex Virus. Muesing, M. A. et al., Cell,48:691-701 (1987); Jones, K. A. et al., Science, 232:755-759 (1986);Gendelman, H. E. et al., Proc. of the Natl. Acad. of Sc., USA,83:9759-9763 (1986); Nabel, G. J. et al., Science (1988); Rando, R. F.et al., Oncogene, 1:13-19 (1987); Mosca, J. D. et al., Nature, 325:67-70(1987). It is doubtful whether the tat-III and Spl proteins areresponsible for an initial induction of HIV expression. Although thetat-III protein functions as a strong positive feedback regulator of HIVexpression, full expression of the tat-III protein appears to depend onNF-kB. Muesing, M. A. et al., Cell, 48:691-701 (1987); Nabel, G. and D.Baltimore, Nature, 326:711-713 (1987). It is unlikely that Spl initiatesHIV expression because it is constitutively active. Dynan, W. S. and R.Tjian, Cell, 32:669-680 (1983). The viral ElA and ICPO gene productsmight lead to induction of HIV expression. This, however, is independentof T-cell activation by antigenic stimulation and of NF-kB, as shown bycotransfection experiments into human T-lymphoma cells of plasmids withan HIV enhancer-controlled CAT gene and plasmids encoding the viralgenes. The increase in CAT activity induced by the viral gene productswas unchanged when the NF-kB binding sites in the HIV enhancer wereinactivated by mutation.

Improved DNA Binding Assay with Enhanced Sensitivity for Identificationof Regulatory Factors

The transcriptional regulatory factors described above were identifiedin extracts of cellular nuclear protein by means of an improved gelelectrophoresis DNA binding assay with enhanced sensitivity. Thisimproved assay is a modification of an original assay based on thealtered mobility of protein-DNA complexes during gel electrophoresis. Inthe improved assay of this invention, the simple alternating copolymer,duplex poly(dI-dC)-poly(dI-dC) was used as the competitor DNA species.The use of this copolymer as competitor resulted in an enhancement ofsensitivity for detection of specific protein-DNA complexes. Theoriginal assay has been extensively employed in equilibrium and kineticanalyses of purified prokaryotic gene regulatory proteins. See, e.g.,Fried, M. and Crothers, D. M., Nucleic Acid Res. 9 6505-6525 (1981);Garner, M. M. and Revzin A., Nucleic Acids Res. 9 3047-3060 (1981). Morerecently it has been used to identify and isolate a protein that bindsto satellite DNA from a nuclear extract of eukaryotic cells (monkeycells). See Strauss, R. and Varshavsky, A., Cell 37 889-901 (1984). Inthe latter study an excess of heterologous competitor DNA (E. coli) wasincluded with the specific probe fragment to bind the more abundant,sequence non-specific DNA binding proteins in the extract.

The assay is performed essentially as described by Strauss andVarshavky, supra, except for the addition of thepoly(dI-dC)-poly(dI-dC). An extract of nuclear protein is prepared, forexample, by the method of Dingnam, J. D. et al., Nucleic Acids Research11:1475-1489 (1983). The extract is incubated with a radiolabelled DNAprobe (such as an end-labeled DNA probe) that is to be tested forbinding to nuclear protein present in the extract. Incubation is carriedout in the presence of the poly(dI-dC)-poly(dI-dC) competitor in apysiological buffer. DNA protein complexes are resolved from (separatedfrom) free DNA probes by electrophoresis through a polyacrylamide gel ina low ionic strength buffer and visualized by autoradiography.

In a preferred embodiment of the method, protein samples (about 10 μgprotein) are incubated with approximately 10,000 cpm (about 0.5 ηg) ofan end-labeled ³² P double-stranded DNA probe fragment in the presenceof about 0.8-4 ηg poly(dI-dC)-poly(dI-dC) (Pharmacia) in a final volumeof about 25 μl. Incubations are carried out at 30° for 30-60 minutes in10 mM Tris HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, 1 mM EDTA. Protein-DNAcomplexes are resolved on low-ionic strength polyacrylamide gels.Samples are layered onto low ionic-strength 4% polyacrylamide gels(0.15×16 cm; acrylamide:bisacrylamide weight ratio of 30:1). Gels arepre-electrophoresed for about 30 min at 11 V/cm in buffer consisting of6.7 mM TrisHCl, (pH 7.5), 3.3 mM NaOAc, and 1 mM EDTA. Buffer isrecirculated between compartments. Gels are electrophoresed at the samevoltage at room temperature, transferred to Whatman 3MM, dried andautoradiographed.

The enhanced sensitivity of the assay of the present invention isevident in the initial work which led to identification of the factorIgNF-A. A radiolabelled SfaNI-SfaNI DNA fragment derived from theupstream region of the MOPC 41 κ light chain gene (FIG. 1) was incubatedwith a nuclear extract of a human B cell line, in the absence or in thepresence of E. coli chromosomal DNA or poly(dI-dC)-poly(dI-dC). Theresulting complexes were resolved from the free fragment byelectrophoresis through a low ionic strength, non-denaturingpolyacrylamide gel and visualized by autoradiography. In the absence ofcompetitor DNA, all of the labeled fragment was retained at the top ofthe gel, probably due to the binding of an excess of nonsequence-specific proteins. With addition of increasing amounts ofeither poly(dI-dC)-poly(dI-dC) or E. coli chromosomal DNA ascompetitors, putative protein-DNA complexes which migrated slower thanthe free fragment were detected. The relative abundance of the majorspecies of complex (B) as well as that of minor species wassignificantly greater in the presence of the alternating copolymercompetitor DNA.

The use of a sensitive gel electrophoresis DNA binding assay inconjunction with the copolymer competitor poly(dI-dC)-poly(dI-dC)facilitated the identification of the regulatory factors describedherein. The simple alternating copolymer probably competes lesseffectively than heterologous DNA sequences for binding of asequence-specific factor, thereby significantly increasing thesensitivity of the assay. The assay has general applicability forelucidation of mammalian gene regulatory proteins.

A further increase in sensitivity in this assay is obtained by the useof small DNA probes (about 100 bp or less) which minimize non-specificbinding interactions in a crude extract. (See Example 1).

Employing this assay, binding competition tests can be performed toanalyze the sequence specificity of protein-DNA interactions. For thispurpose, an unlabeled DNA fragment to be examined for competitivebinding to the protein factor can be added to the incubation mixture ofprotein extract and labeled DNA probe (along with thepoly(dI-dC)-poly(dI-dC)). The disappearance of protein-DNA probecomplex, or its diminishment, indicates that the unlabeled fragmentscompete for binding of the protein factor. In addition, relative bindingaffinity of the protein to a probe sequence can be assessed by examiningthe ability of a competitor to displace the protein at varyingconcentrations.

In conjunction with the competition assays, DNase I footprint analysis(See Galas, D. and Schmitz A., Nucl. Acids Res. 5 3157-3170 (1978) andExample 1) and methylation interference experiments (See, e.g.,Ephrussi, A. et al., Science 227:134-140 (1985) can be used to refineanalysis of the binding domain of the protein factors.

Assessment of the Functional Role of Factors Described Herein inRegulation of Transcription

The functional role of the factors in the regulation of thetranscription can be assessed in several ways. A preferred technique forlymphoid cell factors entails the use of the in vitro transcriptionsystem developed from cells of lymphoid cell lineage. This system isdescribed in detail in the Example 2. The function of a factor can beindirectly assessed in this system by employing as templates fortranscription, nucleotide sequences from which the binding domain of thefactor has been deleted. As has been noted above, deletion of theupstream sequence located between -44 and -79 bp from the cap site ofthe MOPC41 κ gene disrupts transcriptions in this system (This has alsobeen noted in in vivo systems). The deleted region includes the IgNF-Abinding site. This indicates that transcription of the template isdependent upon the factor--binding site and, inferentially, upon thefactor itself.

A direct way to assess the function of the factors is to show thattranscription can be modulated by removal and replacement of the factorin the in vitro transcription system with an appropriate template. Forexample, the intact MOPC41 κ promoter gene can be used as a template inthe in vitro system described and transcription of this template can beassessed in the presence and absence of a factor (for instance, NF-κB, alymphoid specific factor). The factor can be removed from the lymphoidcell extract by chromatographic fractionation and then replaced. If thelevel of transcription is diminished in the absence and restored byreplacement of the factor, a direct indication of the factorsinvolvement in transcription is provided.

In an alternative approach, antisera or monoclonal antibody can beraised against a purified or enriched preparation of the factor. Theantibody can be used to probe for expression of the factor in a libraryof cDNA of cells known to express the factor.

Cloning of Genes Encoding Sequence-Specific DNA Binding Proteins,Particularly Genes Encoding Transcriptional Regulatory Factors

Genes encoding transcriptional regulatory factors can be isolated by anovel method for cloning genes that encode sequence-specific DNA bindingproteins. The method involves screening a library of recombinantexpression vectors for expression of the factor with a DNA probecomprising the recognition (binding) site for the factor. Expression ofthe factor is identified by the presence of complex between the DNAprobe and the expressed binding protein. The approach has generalapplicability to the cloning of sequence-specific DNA binding proteins.

According to the method, an expression library is created by insertingDNA (e.g., cDNA from a cell which expresses the sequence specificbinding protein) into an appropriate expression vector to establish anexpression library. A preferred expression vector is the bacteriophageλgt11 which is capable of expressing foreign DNA inserts within E. coli.See e.g., Young, R. A. and Davis, R. W. in Genetic Engineering:Principles and Techniques, vol 7 (eds Setlow, J. & Hollaender, A.) 29-41(Plenum, New York 1985). Alternatively, plasmid vectors may be used.

The expression library is screened with a binding-site DNA probe. Theprobe comprises the DNA sequence recognized by the binding protein, suchas an appropriate transcriptional regulatory element (e.g., the octameror κ-element). In preferred embodiments, the probe is less than 150 bpin length, to reduce nonspecific binding. The probe can be an oligomerof the binding site. Multiple copies of the site provide for multipleprotein binding to the probe. The DNA probe is generally detectablylabeled DNA. A particularly useful label is ³² P.

In the present method, the binding site probe is incubated with hostcell protein under conditions which allow the probe to complex with theany cognate binding protein expressed in the cell. The formation of suchcomplexes is determined by detecting label associated with the protein.In a preferred mode, the screening is performed by generating a replicaof host cell lysates and by screening the replicated protein with theprobe. For example, when the bacteriophage λgt11 is used, recombinantviruses are plated in arrays onto a lawn of E. coli and a replica of theresulting viral plaques is made by transferring plaque protein onto anappropriate adsorbtive surface (e.g. protein replica filters). Theadsorbed plaque protein is contacted with the probe under conditionswhich permit the formation of complexes between adsorbed protein and theprobe. The replica is then washed to remove unbound probe and thenexamined for associated label. The protein can be examinedautoradiograghically for the presence of label.

In other embodiments, a nonspecific competitor DNA can be used alongwith the recognition site probe, to reduce nonspecific binding to theprobe. Examples of such nonspecific competitor DNA include poly(dI-dC)-poly(dI-dC) and denatured calf thymus DNA. In addition, theprotein-probe complexes can be stabilized covalently for detection, forexample, by uv irradiation

This method of screening for sequence specific binding proteins isdependent, inter alia, upon:

i) the functional expression of the binding domain of the desiredbinding protein in the host cell;

ii) a strong and selective interaction between the binding domain andthe DNA probe; and

iii) a sufficiently high level of expression of the binding protein.

These parameters can be optimimized for different proteins by routineexperimentation. Some factors relevant to such optimization arediscussed in detail in the exemplification of the cloning oftranscriptional regulatory factor NF-κB given below.

Other modes of cloning genes encoding sequence-specific DNA bindingproteins, such as genes encoding transcriptional regulatory factors, maybe used. For example, the factor can be purified chromatographically by,for example, ion exchange, gel filtration and affinity chromatography orcombinations thereof. Once the factor is sufficiently purified, it canbe partially sequenced and from the sequence information,oligodeoxy-nucleotide probes can be made and used to identify the geneencoding the factor in a cDNA library.

Occurrence and Activation of NF-kB and Demonstration of the Role of anNF-kB Inhibitor (IkB)

The following is a description of the occurrence and activation of NF-kBin cells which do not express k immunoglobulin light chain genes (and,in which NF-kB is not evident in either cytoplasmic or nuclearfractions). In particular, the following is a description oflocalization of NF-kB in the cytosolic fraction; of activation of NF-KBin cytosolic fractions by dissociating agents; of redistribution ofNF-kB into the nuclear fraction upon TPA stimulation; of demonstrationof the appearance of NF-KB binding ability; and of the occurrence andcharacterization of an NF-kB inhibitor.

NF-KB Occurrence and Activation in 70Z/3 Cells

NF-KB is Virtually Undetectable in Unstimulated 70Z/3 Cells

To determine where in the cell NF-kB or its inactive precursor arelocated, subcellular fractions from control and TPA-stimulated 70Z/3cells were analyzed for kB-specific DNA-binding activity. Nuclearextracts, cytosolic and postnuclear membrane fractions were analyzed atequal amounts of protein in an electrophoretic mobility shift assay,described in Example 1, followed by fluorography. (Sen, R. and D.Baltimore, Cell, 46:705-716 (1986). The specificity of protein-bindingto a fragment of the kappa light chain enhancer was controlled by usinga fragment with a mutation in the binding motif for NF-kB. This mutationhas been shown to prevent binding of NF-KB. Lenardo, M. et al., Science,236:1573-1577 (1987). Thus, any complexes formed on the wild type, butnot on the mutant fragment, are considered specific for the NF-KB site.

Nuclear extracts from control cells contained very little kb-specificbinding activity. This is in agreement with results reported previouslyby Sen and Baltimore. Sen, R. and D. Baltimore, Cell, 46:705-716 (1986);Sen, R. and D. Baltimore, Cell, 47:921-928 (1986). Similarly, thectyosolic fraction produced only a faint, but specific and reproducible,signal co-migrating with the signal from the nuclear extract. Thefraction containing postnuclear membranes did not exhibit any detectableDNA-binding activity.

Upon treatment of cells with TPA for 30 minutes, the nuclear NF-kBactivity was dramatically increased. Almost no increase of the specificsignal in the cytosolic fraction was observed. The post-nuclear membranefraction gave raise to an apparently kB-specific complex with a mobilityhigher than that formed by nuclear NF-kB. None of the fractions hadinhibitors of binding because added authentic NF-kB was fully recoveredin all fractions, indicating that the results reflect a true activationof binding specificity.

NF-kB is Detectable in the Cytosolic Fraction after Denaturation andRenaturation

To examine whether active NF-kB might be present but masked in fractionsfrom unstimulated 70Z/3 cells, proteins from nuclear extracts andcytosolic fractions of control and TPA-stimulated cells wereprecipitated, denatured by boiling in SDS plus 2-mercaptoethanol andfractionated by electrophoresis through SDS-polyacrylamide gels. 300 ugof protein of nuclear extracts (N) and cytosolic fractions (C) fromcontrol (Co) and TPA-stimulated cells (TPA) were subjected to reducingSDS-polyacrylamide gel electrophoresis. Proteins eluted from differentmolecular weight fractions of the gel (i.e., corresponding toapproximately 70-62 kDa (gel slice No. 6), 62-55 kDa (gel slice No. 7)and 55-48 kDa (gel slice No. 8)) were subjected to a renaturationprotocol after removal of SDS. Hager, D. A. and R. R. Burgess, Anal.Biochem., 109:76-86 (1980) and Example 10. Renatured fractions weretested for kB-specific DNA-binding activity in mobility shift assaysusing wild type and mutant kappa light chain enhancer fragments.DNA-binding reactions were performed with 11 ul of the renaturedfractions in the presence of 80 ng poly(d I-c!) in a final volume of 15ul. Assays with wild type (WT) and mutant (mu) k enhancer fragments wereloaded in adjacent lanes.

In nuclear extracts from TPA-stimulated cells, NF-kB activity wasexclusively found in a molecular weight region of 62-55 kDa. Theefficiency of renaturation of the nuclear NF-kB activity was about onepercent. In nuclear extracts from control cells, much less NF-kBactivity was found in the same molecular weight fraction afterrenaturation. Both the cytosolic fractions from control andTPA-stimulated cells, however, gave rise to a strong NF-kB-specificsignal. The specificity of the signal was shown by several criteria.First, it was only present when the wild type, but not the mutant, DNAfragment was used in mobility shift assays. Second, it was generatedwith protein eluted from the same molecular weight fraction thatcontained authentic nuclear NF-kB. Third, upon mixing, the complexformed by the putative cytoplasmic NF-kB co-migrated exactly in nativepolyacrylamide gels with the complex formed by interaction of thenuclear form of NF-kB with its cognate DNA.

Assuming that NF-kB from the various fractions had a similar recoveryand efficiency of renaturation, the data suggest that significantamounts of NF-kB can be activated in unstimulated 70Z/3 cells bydenaturation, followed by fractionation and renaturation. Furthermore,in unstimulated cells, the in vitro activated NF-kB activity was almostexclusively recovered in the cytosolic fraction.

The subcellular distribution of two noninducible DNA-binding proteins,NF-uE3 and the octamer-binding protein were also examined in mobilityshift assays, in order to determine whether other DNA-binding factorsalso partition into cytoplasmic fractions. Sen, R. and D. Baltimore,Cell, 46:705-716 (1986); Singh, H. et al., Nature, 319:154-158 (1986);and Staudt, L. M. et al., Nature, 323:640-643 (1986). The vast majorityof both DNA-binding activities was found in nuclear extracts; cytosolicand postnuclear membrane fractions contained only very littleactivities. No significant change in the complex formation by the twofactors was observed when fractions from control and TPA-stimulatedcells were compared. Thus, although subcellular fractionation canproduce artificial redistribution of proteins, the fractions used inthis study do well reflect nuclear localization of a number ofDNA-binding proteins.

NF-kB in the Cytosolic Fraction Can be Activated by Dissociating Agents

The ability to reveal cytosolic NF-kB by simply denaturation andrenaturation suggested that NF-kB might be bound to an inhibitor andtherefore several compounds that might dissociate protein complexes weretested for their ability to directly activate kB-specific DNA-bindingactivity in fractions of 70Z/3 cells. The cytosolic fraction fromunstimulated cells and, as a control, the nuclear extract fromTPA-treated cells were incubated with the compounds prior toelectrophoretic separation of the protein-DNA complexes. Incubation ofthe cytosolic fraction with 0.2% sodium desoxycholate (DOC) (in thepresence of 0.2% NP-40) resulted in the activation of DNA-bindingactivity. The induced complex had the same mobility in native gels asthe one formed by nuclear NF-kB. It appeared to be specific for the kBsite of the kappa light chain enhancer because it was not formed whenthe mutant fragment was used in the mobility shift assay. Higherconcentrations of DOC led to the inactivation of the newly activatedkB-binding activity as well as of the authentic nuclear factor.

DOC can be sequestered out of a solution by the addition of excessnonionic detergent, presumably by inclusion of the DOC into micellesformed by the nonionic detergent. When treatment of the cytosolicfraction with up to 0.8% DOC was followed by the addition of 1% of thenonionic detergent NP-40, a quite efficient activation of the cytosolickB-binding activity was achieved. The DNA-binding activity of in vivoactivated NF-kB from nuclear extracts was not significantly increased atlow concentrations of DOC. Elevated concentrations of DOC showedinhibitory effects on the DNA-binding activity of TPA-activated NF-kBthat paralleled those observed for the in vitro activated kB-bindingactivity.

A partial activation of the cytosolic kB-binding activity was observedafter treatment of the cytosolic fraction with 27% formamide followed bydilution. With the further addition of 0.2% DOC--a condition that alonealso leads only to partial activation--a very potent activation wasobserved. A titration showed that formamide and DOC activated in asynergistic manner. The DNA-binding activity of in vivo activated NF-kBfrom nuclear extracts was not enhanced by any of the treatments. On thecontrary, partial inhibition of DNA-binding of NF-kB was observed undersome conditions.

No in vitro activation of NF-kB was achieved by treatment withguanidinium hydrochloride (between 0.3 and 3M), urea (between 0.5 and5M), and SDS (between 0.1 and 1%), in the presence of 0.2% NP-40).Exhaustive dialysis of the cytosolic fraction using dialysis membraneswith a cut-off of 25 kDa did not lead to an activation of DNA-bindingactivity. In the dialyzed fraction, NF-kB-activity could still beefficiently induced by formamide/DOC treatment, suggesting that nofreely diffusible cofactors smaller than 25 kDa were required for the invitro activation.

TPA Stimulation Causes Redistribution of NF-kB into the Nuclear Fraction

To examine whether the form of NF-kB detected after in vitro activationin the cytosolic fraction could quantitatively account for the NF-KBfound in nuclear extracts after TPA stimulation of cells, subcellularfractions of 70Z/3 cells were reinvestigated in mobility shift assaysafter treatment with formamide and DOC using equal cell-equivalents ofsubcellular fractions. Equal cell-equivalents of nuclear extracts (N)and cytosolic (C) and post-nuclear membrane fractions (P) from control(Co) and TPA-stimulated cells (TPA) were left untreated or subjected toa formamide/desoxycholate treatment. This treatment was preferred overthe DOC/NP-40 chase treatment because it gave a higher resolution ofbands in mobility shift assays. DNA-binding reactions were performed inthe presence of 3.2 ug poly(d I-C!) using 4.4 ug of protein from nuclearextracts, 8.8 ug of protein from cytosolic fractions or 2.2 ug ofprotein from postnuclear membrane fractions (all in 4 ul buffer D(+)).Fluorograms of native gels are shown. The specificity of protein-DNAcomplexes was controlled using wild type (kB wt) and mutant kappaenhancer fragments (kB mutant. The filled arrowhead indicates theposition of kB-specific protein-DNA complexes and the open arrowhead thepositions of unbound DNA-fragments.

Densitometric scanning of fluorograms showed that in control cells, morethan 92% of the total cellular kB-specific DNA-binding activity wasrecovered in the cytosolic fraction following treatment with formamideand COD. In TPA-stimulated cells, 80% of the kB-specific DNA-bindingactivity was found in nuclear extracts. The remaining activity waslargely recovered in the cytosolic fraction. All DNA-binding activitiesdescribed were specific for the kB site, as shown by their absence whenthe mutant kappa enhancer fragment was used in the mobility shiftassays.

When the total cellular NF-kB activity that was activated in vitro incontrol cells was compared to the total cellular activity found inTPA-stimulated cells after the same treatment, virtually identicalamounts of activity were observed. The equal amounts of NF-kB activityfound in control and TPA-treated cells suggest that the treatment withformamide and DOC resulted in the complete conversion of an inactiveprecursor of NF-kB into a form of NF-kB with high DNA-binding affinity.Furthermore, these results provide evidence for a TPA-inducibletranslocation of NF-kB from the cytosol into the nucleus.

NF-kB Occurrence and Activation in HeLa Cells

NF-kB activity can also be induced in HeLa cells after TPA treatment, asshown by the appearance of a kB-specific DNA-binding activity in nuclearextracts. Sen, R. and D. Baltimore, Cell, 47:921-928 (1986). Therefore,induction of NF-kB in the cytosolic fraction of HeLa cells was tested bytreatment with formamide and DOC. To equal cell-equivalents offractions, 17% formamide was added and diluted to 10% by the addition ofthe DNA-binding reaction mixture containing 4 ug poly(d I-C!). DOC wasthen added to a final concentration of 0.6% to give a reaction volume of20 ul. Assays contained either 1.35 ug of protein from nuclear extracts,9 ug of protein from the cytosolic fractions of 0.9 ug of protein fromthe postnuclear membrane fractions (all in 10 ul buffer D(+)).

Redistribution of NF-kB activity in the subcellular fractions upon TPAstimulation of cells, was also assessed, using the procedure describedfor 70Z/3 cells. Mobility shift assays were performed with equalcell-equivalents of the subcellular fractions. Because HeLa cells hadabout ten times as much cytosolic protein as nuclear protein--as opposedto the 2:1 ratio in 70Z/3 cells--the use of equal cell-equivalents offractions gave very different quantitative results from those obtainedwith equal amounts of protein. Without any treatment, only traces of akB-specific DNA-binding activity were detected in the nuclear andcytosolic fractions of HeLa cells and no activity was observed in thepostnuclear membrane fraction. Upon TPA stimulation of cells under thesame conditions as for 70Z/3 cells, NF-kB activity was stronglyincreased in the nuclear extract. Also, in the cytosolic fraction, asignificant increase of NF-kB activity was found. This was not anartifact of fractionation because the activity of AP-1, another nuclearfactor (Lee, W. et al., Cell, 49:741-752 (1987), was highly enriched innuclear extracts and almost not detectable in the cytosolic fraction ofHeLa cells before and after TPA stimulation.

The treatment of control fractions of HeLa cells with formamide and DOCrevealed large amounts of kB-specific DNA-binding activity in thecytosolic fraction. The concentrations of formamide and DOC required foran optimal in vitro activation of NF-kB in HeLa cells were differentfrom those required for 70Z/3 cells; less formamide and more DOC wasneeded. All DNA-binding activities described were specific for thekB-binding site in the kappa enhancer fragment.

Almost no activity was detected in the HeLa nuclear extract and thepostnuclear membrane fraction after in vitro activation. Large amountsof NF-kB activity could still be activated in the cytosolic fraction ofTPA-stimulated HeLa cells. This suggests that in vivo in HeLa cells--ascontrasted to 70Z/3 cells--only a minor portion of the total cellularNF-kB is activated upon a TPA stimulus. The NF-kB activity informanide/DOC-treated nuclear extracts of TPA-stimulated cells was less,compared to untreated nuclear extracts, reflecting a partial inhibitionof the DNA-binding activity of in vivo activated NF-kB. As in 70Z/3cells, the total cellular NF-kB activity in HeLa cells, as revealedafter in vitro activation, remained constant before and after TPAtreatment of cells. These data imply that NF-kB is activated by the samemechanism in HeLa cells as it is in the pre-B cell line 70Z/3. However,in HeLa cells, TPA is much less complete in its activation than it is in70Z/3 cells.

NF-kB Occurrence and Activation in Other Cell Types

NF-kB occurrence and activation in several additional cell types,including two T cell lines (H9, Jurkat) and fibroblasts, and in tissues,including human placenta and mice kidney, liver, spleen, lung, muscleand brain, were also assessed, as described above. In each case, NF-kBin a DOC-activatable form was shown to be present in the cytosolicfraction.

Appearance of Binding Activity

Results described above suggested that the appearance of bindingactivity may be due to separation of NF-kB from an inhibitor. Sizefractionation and denaturing agents were both shown to be capable ofseparating NF-kB from such an inhibitor, which was apparently of lowmolecular weight. This provides a reasonable explanation for how NF-kBis induced in pre-B cells, HeLa cells and other inducible cells, such asT cells.

Whether the DOC-dependence of cytosolic NF-kB results from itsassociation with an inhibitor, was investigated by probing for activityin cytosolic fractions that would specifically prevent DNA binding toNF-kB in electrophoretic mobility shift assays (EMSA). This workdemonstrated the existence of a protein inhibitor, called IkB, incytosolic fractions of unstimulated pre-B cells, that can convert NF-kBinto an inactive DOC-dependent form by a reversible, saturable, andspecific reaction. The inhibitory activity becomes evident afterselective removal of the endogenous cytosolic NF-kB under dissociatingconditions, suggesting that NF-kB and IkB were present in astoichiometric complex. Enucleation experiments showed that the complexof NF-kB and IkB is truly cytoplasmic. The data are consistent with amolecular mechanism of inducible gene expression by which a cytoplasmictranscription factor-inhibitor complex is dissociated by the action ofTPA, presumably through activation of protein kinase C. The dissociationevent results in activation and apparent nuclear translocation of thetranscription factor. It would appear that IkB is the target for theTPA-induced dissociation reaction. The following is a description ofthis investigation, which is described in greater detail in Example 12.

Separation of an Inhibitor from NF-kB

Cytosolic fractions from unstimulated 70Z/3 pre-B cells were examinedfor an activity that would impair the DNA binding activity of addedNF-kB in an EMSA. Baeuerle, P. A., and D. Baltimore, Cell 53: 211(1988). Increasing amounts of cytosol from unstimulated cells did notsignificantly influence the formation of a protein-DNA complex betweenNF-kB and a k enhancer fragment. This indicated the absence of freeinhibitor, presumably because all of it is complexed with endogenousNF-kB. DNA-cellulose was used to selectively remove the endogenous NF-kBfrom DOC-treated cytosol, in an attempt to liberate the inhibitor.Almost all NF-kB was present in a DOC-dependent form prior to DOCactivation and chromatography. In the presence of excess DOC, about 80%of the NF-kB activity was retained by DNA-cellulose, most of whicheluted from the DNA-cellulose between 0.15 and 0.35M NaCl. The NF-kBactivity eluting at high salt was detectable in mobility shift assays inthe absence of excess DOC, indicating that NF-kB had been separated froman activity that caused its DOC-dependent DNA binding activity. Incontrast, the small percentage of NF-kB activity contained in thewashings was still dependent on DOC. These results show that affinitychromatography is sufficient to convert DOC-dependent NF-kB precursorinto DOC-independent active NF-kB, similar to that found in nuclearextracts from TPA-stimulated cells.

The flow-through fraction from the DNA-cellulose was assayed for anactivity that, after neutralization of DOC by non-ionic detergent, wouldinactivate added NF-kB from the 0.2M NaCl-fraction from nuclear extractsof TPA-stimulated cells. Increasing amounts of cytosol from which theendogenous NF-kB was removed inhibited the formation of an NF-kB--DNAcomplex as monitored by EMSA. DOC-treated cytosol that was not passedover DNA-cellulose had no effect, even if cells had been treated withTPA. The fact that, after DNA-cellulose chromatography of DOC-treatedcytosol, both DOC-independent NF-kB and an inhibitory activity wereobserved made it reasonable to believe that NF-kB had been separatedfrom an inhibitor. This inhibitor is referred to as IkB.

IkB Characterization

IkB fractionates as a 60 to 70 kD protein. The flow-through fractionfrom the DNA-cellulose column was subjected to gel filtration throughG-200 Sephadex and the fractions were assayed for an activity that wouldinterfere with the DNA binding activity of added NF-kB contained in anuclear extract from TPA-stimulated 70Z/3 cells. The 67 kD fraction hadthe highest activity: it virtually completely prevented interaction ofNF-kB and DNA. In fractions from a G-75 Sephadex column, no additionalinhibitor of low molecular size was detectable indicating that NF-kB wasinactivated by a macromolecule of defined size. No significantinhibitory activity could be demonstrated after gel filtration of aDNA-cellulose flow-through of DOC-treated cytosol from TPA-stimulated70Z/3 cells, implying that TPA treatment of cells inactivated IkB.

The inhibitor fraction was treated with trypsin to test whether IkB is aprotein. Tryptic digestion was stopped by the addition of bovinepancreas trypsin inhibitor (BPTI) and samples were analyzed for NF-kBinhibition. Trypsin treatment interfered with the activity of IkB, asshown by the complete inability of the treated sample to inhibit NF-kBactivity. Trypsin that had been treated with BPTI had no effect,demonstrating that the inactivation of IkB was specifically caused bythe proteolytic activity of trypsin. It appears that IkB requires anintact polypeptide structure for its activity.

The cytosolic complex of IkB and NF-kB showed an apparent size of about120 to 130 kD, both after gel filtration and after sedimentation througha glycerol gradient. For both methods, cytosol from unstimulated cellswas analyzed under non-dissociating conditions. NF-kB was activated infractions by either DOC or formamide prior to analysis by EMSA.Baeuerle, P. A. and D. Baltimore, Cell, 53:211 (1988). The specificityof complexes was tested with a mutant DNA probe. Lenardo, M. et al.,Science, 236:1573 (1987). The apparent release of a 60 to 70 kDinhibitory protein from the cytosolic NF-kB precursor, its sedimentationvelocity in glycerol gradients, and its size seen by gel filtrationsuggest that the inactive NF-kB precursor is a heterodimer composed of a55 to 62 kD NF-kB molecule and a 60 to 70 kD IkB molecule. Nuclear NF-kBwas found to cosediment with the cytosolic complex of IkB and NF-kB.Native gel electrophoresis, a method that allows resolution of sizedifferences of protein-DNA complexes, provided evidence that the 120 kDform of nuclear NF-kB seen in glycerol gradients comes from theformation of a homodimer. Hope, I. A. and K. Struhl, EMBO J., 6:2781(1987). By these interpretations, activation of NF-kB would include anadditional step (i.e., formation of a NF-kB homodimer). This isconsistent with the observation that the protein-DNA complexes formedwith in vitro-activated NF-kB have the same mobility in native gels asthose formed with nuclear NF-kB. Baeuerle, P. A. and D. Baltimore, Cell,53:211 (1988).

The inactivation of NF-kB by IkB is reversible, saturable and specific.Incubation with the inhibitor fraction can inhibit the DNA bindingactivity of NF-kB by more than 90%. Treatment of a duplicate sample withDOC after the inhibition reaction reactivated 66% of the added NF-kBactivity. This showed that a DOC-dependent form of NF-KB can bereconstituted in vitro by the addition of a fraction containing IkB tonuclear NF-kB. The incomplete activation of NF-kB by DOC might be due tothe DOC-neutralizing effect of non-ionic detergent which was stillpresent in the sample from the preceding inhibition reaction.

A titration and kinetic analysis showed that IkB stoichiometricallyinteracts with NF-kB (FIG. 16). Increasing amounts of inhibitor fractionwere added to an excess amount of NF-kB and incubated for 20 or 60minutes. After the DNA binding reaction, NF-kB-DNA complexes wereseparated on native gels and quantified by liquid scintillationcounting. The relationship between amount of IkB fraction added andextent of inhibition was linear. The amount of NF-kB inactivated after20 minutes of incubation was not increased after 60 minutes (FIG. 16).These kinetics were probably not the result of a rapid decay of acatalytically active inhibitor because the fractions were incubatedprior to the reaction. The data are consistent with rapid formation ofan inactive complex by addition of IkB to NF-kB. The fraction containingIkB does not appear to catalytically or covalently inactivate NF-kB:neither the reversibility nor the kinetics support such a model.

IkB was tested for its influence on the DNA binding activity of otherdefined nuclear factors (FIG. 7). These factors were contained innuclear extracts that had essentially no active NF-kB, which otherwisecould have inactivated IkB by complex formation. The DNA bindingactivity of H2TF1, a transcription factor thought to be related toNF-kB, was not affected by the inhibitor fraction. Baeuerle, P. A. etal., unpublished observation). Ubiquituous and lymphoid-specificoctamerbinding proteins (OCTA) (Sive, H. L. and R. G. Roeder, Proc.Natl. Acad. Sci. USA, 83:6382 (1986) and Staudt, L. M. et al., Nature,323:640 (1986)) were unaffected in their DNA binding activities, as weretwo E-box binding factor, NF-μE1 (Weinberger, J. et al., Nature, 322:846(1986)) and NF-kE2 (Lenardo, M. et al., Science, 236:1573 (1987)),interacting with μ heavy chain and k light chain enhancers,respectively. AP-1, another TPA-inducible transcription factor (Lee, W.et al., Cell, 49:741 (1987); Angel, P. et al., Cell, 49:729 (1987)),also showed equal complex formation after incubation in the presence andabsence of the inhibitor fraction. Furthermore, none of the undefinedDNA binding activities seen in the EMSA showed any inactivation by IkB.These results show that IkB is a specific inhibitor of the DNA bindingactivity of NF-kB.

In vivo activated NF-κB is responsive to IkB. IkB prepared from themouse pre-B cell line 70Z/3 was tested for inactivation of NF-kBcontained in nuclear extracts from other cell lines. Human NF-kBcontained in nuclear extracts from TPA-stimulated HeLa cells and H-9T-lymphoma cells was efficiently inactivated. When excess amounts of thevarious NF-kB activities were used in the inhibitor assay, the extent ofreduction of NF-kB activities by a fixed amount of IkB was very similar,as quantified by liquid scintillation counting. NF-kB from nuclearextracts of TPA-stimulated Madin-Darby bovine kidney (MDBK) cells wasalso inactivated suggesting that the control of NF-kB activity by IkB isconserved among different mammalian species.

NF-kB is constitutively active in cell lines derived from mature Bcells. Sen, R. and D. Baltimore, Cell, 46:705 (1986). Nuclear extractsfrom the mouse B cell line WEHI 231 were tested in the inhibitor assayto examine whether NF-kB has undergone a modification in those celllines that prevented its inactivation by IkB. NF-kB from B cells was asefficiently inactivated as NF-kB from pre-B cells, suggesting that NF-kBis not stably modified in B cells (or in other cells after TPAstimulation) in such a way that it cannot respond to inactivation byIkB.

The NF-kB--IkB complex is present in enucleated cells. The NF-kB--IkBcomplex shows a cytosolic localization on subcellular fractionation.This procedure may, however, cause artifacts. Hypotonic lysis of cellsmay result in partitioning of nuclear proteins into the cytosol,especially, when they are not tightly associated with nuclearcomponents. Li, J. J. and T. J. Kelly, Proc. Natl. Acad. Sci, USA,81:6973 (1984). Detection of the complex of IkB and NF-kB in enucleatedcells was attempted. Enucleation is performed with living cells at 37°C. and should therefore not interfere with active nuclear import ofproteins, which is ATP-dependent and blocked at low temperature.Prescott, D. M. and J. B. Kirkpatrick, In: Methods Cell Biol., D. M.Prescott, ed. (Academic Press, New York, 1973), p. 189; Newmeyer, D. D.and D. J. Forbes, Cell, 52:641 (1987); Richardson, W. D. et al., Cell,52:655 (1988).

Using cytochalasin B-treated HeLa cells, an enucleation efficiency ofabout 90% was obtained. Enucleated and cytochalasin B-treated completecells were incubated in the absence and presence of TPA, solubilized bydetergent and proteins were extracted with high salt. Because of thesmall number of cells analyzed, this procedure is different from thestandard one. Total cell extracts were analyzed for NF-kB specific DNAbinding activity by EMSA. In both enucleated and complete cells, similaramounts of NF-kB activity were found after TPA stimulation. The activitywas specific for NF-kB because it was not observed with a mutant kenhancer fragment. Lenardo, M. et al., Science, 236:1573 (1987); Theseresults suggest that TPA-inducible NF-kB in HeLa cells is predominantlycytoplasmic because it was still present in enucleated cells. The NF-kBactivity seen under control conditions was most likely activated by thelysis conditions used because it was also observed in extracts from HeLacells that were not treated with cytochalasin B, but not in fractionsobtained after hypotonic lysis. Baeuerle, P. A. and D. Baltimore, Cell,53:211 (1988). It was still evident, however, that TPA could activateNF-kB in enucleated cells.

After treatment with DOC, total extracts from complete and enucleatedcontrol cells showed about a 2-fold increase in the amount of NF-kBactivity. The demonstration of DOC-activatable NF-kB in enucleatedcells, as well as the presence of similar amounts of total NF-kB inenucleated and complete cells, shows that a substantial amount of thetotal cellular NF-kB--IkB complex was cytoplasmic. In contrast to NF-kB,most of the DNA binding activity of AP-1, a bona fide nuclear protein,was apparently lost by enucleation of cells. Lee, W. et al., Cell,49:741 (1987); Angel, P. et al., Cell, 49:729 (1987).

Mechanism of NF-kB activation

Thus, it has been shown that the NF-kB nuclear transcription factorexists in unstimulated pre-B cells in a cytoplasmic complex with aspecific inhibitory protein, IκB. In this complex, NF-kB does notexhibit DNA binding activity in EMSA and partitions upon subcellularfractionation into the cytosol. The complex is apparently a heterodimerconsisting of about a 60 kD NF-kB molecule and a 60 to 70 kD IkBmolecule. Upon TPA stimulation of cells, or after treatment withdissociating agents in vitro, the NF-kB--IkB complex dissociates. Thisreleases NF-kB, which appears now to form a homodimer and cantranslocate into the nucleus. Whether dimerization is required foractivation of NF-kB is not known.

The inhibitory effect of IkB on the DNA binding activity and nuclearlocalization properties of NF-kB appears to arise from a simple physicalaffinity of the two proteins. The complex freely dissociates and thecomponents readily associate under in vitro conditions. Even in vivo,dissociation by short-term TPA treatment and reassociation afterlong-term TPA treatment is evident. The latter presumably results fromthe degradation of protein kinase C after TPA activation and impliesthat NF-kB can move back to the cytoplasm after being active in thenucleus.

The effect of TPA appears to involve an alteration of IkB, but not ofNF-kB. After TPA stimulation, no active IkB was found--implying itsalteration--while the nuclear NF-κB remained sensitive to unmodified IkBwhen tested in vitro. Whether inactive IkB can be regenerated isunclear; in experiments using cycloheximide (Baeuerle, P. A. et al.,Cold Spring Harbor Symp. Quant. Biol., 53, In Press), irreversible lossof IkB activity was the only demonstrable effect after 8 hours of TPAtreatment. Given the ability of TPA to activate protein kinase C, it isa reasonable hypothesis that direct or indirect phosphorylation of IkBresults in its dissociation from NF-kB.

It had previously been found that the NF-kB--IkB complex is recovered inthe cytosol. It is now shown directly that the complex is not removedfrom the cell by enucleation and, therefore, is truly cytoplasmic.Welshons, W. V. et al., Nature, 307:747 (1984). Because active proteinkinase C is bound to the plasma membrane (Kraft, A. S. et al., J. Biol.Chem., 257:13193 (1983); Wolf, M. et al., Nature, 317:546 (1985);Kikkawa, U. and Y. Nishizuka, Ann. Rev. Cell. Biol., 2:149 (1986)), itbecomes increasingly attractive to suggest that the cytoplasmic complexinteracts in the cytoplasm (maybe near the plasma membrane) with proteinkinase C and the liberated NF-kB carries the signal from cytoplasm tonucleus. Under a number of conditions, active NF-kB is found in thecytoplasm. This fact and the reversibility of NF-kB activation in vivosuggests that the protein may freely move in and out of the nucleus,bringing to the nucleus information reflecting the cytoplasmicactivation state of protein kinase C and possibly of other signallingsystems.

The response of NF-kB to activated protein kinase C occurs apparentlyindirectly through modification and subsequent release of associatedIkB. The inducibility of NF-kB by TPA is thus dependent on the presenceand state of activity of IkB. Changes in amount or activity of IkBshould therefore influence the TPA inducibility of NF-kB. NF-kB canindeed exist not only in TPA-inducible but also in constitutively activeform (e.g., in mature B cells; Sen, R. and D. Baltimore, Cell, 46:705(1986). Because constitutive NF-kB from B cells is still responsive toIkB in vitro, it is thought that IkB, and not NF-kB, is altered duringdifferentiation of pre-B into B cells.

IkB is apparently unstable when not complexed with NF-kB. This issuggested by the absence of excess active inhibitor in the cytosol fromunstimulated cells. In a situation where the production of new inhibitoris impaired, the decay of occasionally released inhibitor could activateNF-kB. This would explain the partial activation of NF-kB seen aftertreatment with the protein synthesis inhibitors cycloheximide andanisomycin. Sen R. and D. Baltimore, Cell, 47:921 (1987). Thedemonstration of a specific inhibitory protein of NF-kB and theinterpretation that cycloheximide treatment can activate NF-kB,presumably because cells become depleted of inhibitor, suggest that IkBis the putative labile repressor of k gene expression (Wall, R. et al.,Proc. Natl. Acad. Sci. USA, 83:295 (1986)) and of NF-kB activity. Sen,R. and D. Baltimore, Cell, 47:921 (1987).

As a result of the work described herein, the IκB gene is now available,as is IκB itself, antibodies specific for the IκB gene-encoded product,and probes which include all or a portion of the IκB gene sequence. Alsoavailable are methods of using the IκB gene, the encoded protein andIκB-specific antibodies for such purposes as identifying and isolatingother IκB genes, IκB "like" genes, and IκB-encoded products. AlteringNF-κB activity and altering NF-κB-mediated gene expression. Inparticular, it is now possible, through the method of the presentinvention, to block or inhibit NF-κB passage into the nucleus of cellsin which it occurs and, thus, block (partially or totally) binding ofNF-κB to NF-κB binding sites on genes which include such recognitionsites. Such a method is useful for altering expression of genes which ismediated by NF-κB; such genes include cellular genes (e.g., cytokinegenes) and genes introduced into host cells (e.g., viral genes, such ascytomegalovirus gene, HIV-1 genes (e.g., the tax gene), and the SV40gene). This method of altering NF-κB-mediated gene expression is useful,for example, for inhibiting viral gene expression in infected cells,such as in an individual infected with the HIV-1 or cytomegalovirus.

The IκB gene and the encoded IκB protein can be used to negativelyregulate NF-κB activity in cells. For example, the IκB gene can beincorporated into an appropriate vector (e.g., a retroviral vector orcapable of expressing the IκB gene and introduced into cells in whichNF-κB activity is to be inhibited (partially or totally). For example, avector capable of expressing IκB can be introduced into HIV-1 infectedcells (e.g, T cells) in order to inhibit HIV-1 gene expression andactivity in the cells. IκB expressed in the cells binds NF-κB (e.g.,free NF-κB such as that released from its inactive complex with Iκ-β)and limits its ability to act as a messenger by inhibiting itstranslocation into the nucleus. For this purpose, all or a portion ofthe IκB-encoding DNA or DNA encoding an IκB-like protein is used. If aportion is used, it must encode at least that region of the IκB (orother rel-associated protein) molecule sufficient to bind NF-κB andprevent it from passing into the cell nucleus. The IκB-encoding DNA orDNA encoding an IκB-like protein used can be obtained from a source inwhich it naturally occurs, can be produced by genetic engineering orrecombinant techniques or can be synthesized using known chemicalmethods. For convenience, DNA from all three types of sources isreferred to herein as "essentially pure". The DNA used can have all or aportion of the DNA sequence of clone MAD-3, all or a portion of pp40 orall or a portion of another sequence which encodes a rel-associated orIκB-like protein capable of inhibiting NF-κB. In a similar manner, DNAencoding a rel-associated or IκB-like protein can be introduced intocells to inhibit a rel-related protein other than NF-κB.

Cells in which IκB (or other rel-associated protein) is to be expressedin this manner to inhibit NF-κB (or other rel-related protein) can beremoved from the body, the IκB-expressing vector can be introduced,using known methods, and the resulting cells, which contain theIκB-expressing vector, then reintroduced into the body. For example,T-cells or bone marrow cells can be removed from an HIV-1 infectedindividual, IκB-expressing vectors can be introduced into them, and theycan then be replaced in the individual. Alternatively, the expressionvector containing IκB-encoding DNA can be introduced into an individual,using known techniques, by any of a variety of routes, such asintramuscular, intravenous, intraperitoneal administration. IκB itself(or other rel-associated protein) can also be introduced into cells toinhibit NF-κB (or other rel-related protein). The entire IκB molecule ora portion sufficient to bind NF-κB and prevent its passage into thenucleus can be used for this purpose. IκB or an appropriate IκB portioncan be obtained from naturally-occurring sources, can be produced usingknown genetic engineering methods or, particularly in the case of an IκBportion, can be synthesized chemically. For convenience, proteins (orportions thereof) of all three types are referred to herein as"essentially pure".

Uses of Genes Encoding Transcriptional Regulatory Factors, the EncodedFactors and Related Products

The genes encoding positive transcriptional regulatory factors provide ameans for enhancing gene expression. Lymphoid-specific factors involvedin positive regulation of Ig gene transcription provide a method forenhancing immunoglobulin production in lymphoid cells. Lymphoid cells,such as monoclonal antibody-producing hybridomas or myelomas, can betransfected with multiple copies of a gene encoding a regulatory factoryto induce greater production of Ig. For this purpose, the gene encodinga regulatory factor can be linked to a strong promoter. In addition, theconstruct can include DNA encoding a selectable marker. Multiple copiesof the contruct can be inserted into the cell, using known transfectionprocedures, such as electroporation. The cell can be transfected withmultiple regulatory factors, including constitutive factors; this isparticularly useful in the case of factors determined to act inconjunction, possibly synergistically. Amplification of genes encodingtranscriptional regulatory factors in this manner results in enhanced orincreased production of the regulatory factors and, consequently,production of immunoglobulin is enhanced in these cells.

The present invention also relates to a method for transientlyexpressing a gene product in a eukaryotic cell, in which theinducibility of the NF-kB factor is used to advantage. This phenomenoncan be exploited to provide for the transient overexpression of a geneproduct produced by a transfected gene in a eukaryotic cell at a chosentime.

According to the method of this invention, a gene of interest is placedunder influence of the κ enhancer sequence containing the binding sitefor NF-kB (i.e., the entire enhancer sequence or a portion containing atleast the NF-kB site). The κ-enhancer sequence is linked to a structuralgene of interest to provide a gene inducible by NF-kB. A gene constructis thus provided comprising 1) a κ-enhancer sequence or a portion of theκ enhancer sequence containing at least the sequence to which the factorNF-kB binds; 2) a promoter; and 3) a structural gene of interest.

Conventional recombinant DNA techniques can be used to prepare theconstruct. The κ enhancer sequence can be obtained from lymphoid cellswhich express the κ-light chain. The κ enhancer can also be obtainedfrom clones containing the sequence. The construct can be prepared in orinserted into a transfection vehicle such as a plasmid.

The structural gene can be any gene or gene segment which encodes auseful protein for which transient overexpression is desired. Suchproteins are, for example, those that are damaging to cells whenproduced constitutively. The structural gene can be used with itsendogenous promoter or other eukaryotic promoter.

Cells for transfection can be any eukaryotic cells used for theexpression of eukaryotic proteins. Transfection procedures, such as thecalcium precipitation technique are well known in the art.

At the desired time, the transfected cells can be stimulated with theappropriate inducer in an amount sufficient to induce production ofNF-kB. The preferred inducer is a phorbol ester which acts rapidly anddirectly to activate protein kinase C and induces production of NF-kB.If the transfected cell is a lymphoid cell (e.g., B cell) responsive toa mitogen such as LPS or PHA the mitogen may be used alone or incombination with phorbol ester.

Genes encoding transcriptional regulatory factors can be modified for avariety of purposes, such as to encode factors with activity equivalentto the naturally-occuring factor, factors with enhanced ability toregulate transcription (e.g., to cause enhanced transcription of genes,relative to transcription resulting from regulation by or the effects ofthe normal/unmodified factor), or factors with decreased ability toregulate transcription. This can be carried out, for example, bymutagenesis of factor-encoding DNA or by producing DNA (e.g., byrecombinant DNA methods or synthetic techniques) which encodes amodified or mutant transcriptional regulatory factor (i.e., atranscriptional regulatory factor with an amino acid sequence differentfrom the normal or naturally-occurring transcription factor amino acidsequence). These modified DNA sequences and encoded modified factors areintended to be encompassed by the present invention.

The gene encoding IgNF-b, for example, has been cloned and sequenced andthe nucleotide sequence is shown in FIG. 11A. For the various utilitiesdiscussed below, the modified nucleotide sequence can be obtained eithernaturally (e.g., polymorphic variants) or by mutagenesis to yieldsubstantially complementary sequences having comparable or improvedbiological activity. Fragments of the sequence may also be used. Thisinvention encompasses nucleic acid sequences to which the sequence ofFIG. 11A hybridizes in a specific fashion.

In addition, the DNA binding domain of the factors, which is responsiblefor the binding sequence-specificity, can be combined with different"activators" (responsible for the effect on transcription) to providemodified or hybrid proteins for transcriptional regulation. For example,with recombinant DNA techniques, DNA sequences encoding the bindingdomain can be linked to DNA sequences encoding the activator to form agene encoding a hybrid protein. The activator portion can be derivedfrom one of the factors or from other molecules. The DNA binding regionof the hybrid protein serves to direct the protein to the cognate DNAsequence. For example, in this way, stronger activators of RNApolymerases can be designed and linked to the appropriate DNA bindingdomain to provide for stronger enhancement of transcription.

DNA probes for the genes encoding the regulatory factors can be used todetermine the presence, absence or copy number of regulatoryfactor-encoding genes or to identify related genes, by hybridizationtechniques or a polymerase chain amplification method (e.g., PCR). Theability to detect and quantify genes encoding transcriptional regulatoryfactors can be used in diagnostic applications, such as to assessconditions relating to aberrant expression of a regulatory factor. Cellscan be typed as positive or negative for the occurrence of a particulargene and, in addition, can be analyzed for the copy number of the gene.The DNA probes are labeled DNA sequences complementary to at least aportion of a nucleic acid encoding a transcriptional regulatory factor.The labeled probe is contacted with a sample to be tested (e.g., a celllysate) and incubated under stringent hybridization conditions whichpermit the labeled probe to hybridize with only DNA or RNA containingthe sequence to which the probe is substantially complementary. Theunhybridized probe is then removed and the sample is analyzed forhybridized probe.

The DNA probes can also be used to identify genes encoding relatedtranscriptional regulatory factors. For this purpose, hybridizationconditions may be relaxed in order to make it possible to detect relatedDNA sequences which are not completely homologous to the probe.

Antibodies can be raised against the transcriptional regulatory factorsof this invention. The antibodies can be polyclonal or monoclonal andthey can be used as diagnostic reagents in assays to determine whether afactor is expressed by particular cells or to quantitate expressionlevels of a factor.

A gene encoding a transcriptional regulatory factor can also be used todevelop in vivo or in vitro assays to screen for agonists or antagonistsof a factor-encoding gene or of the factor encoded by the gene. Forexample, genetic constructs can be created in which a reporter gene(e.g., the CAT gene) is made dependent upon the activity of afactor-encoding gene. These constructs introduced into host cellsprovide a means to screen for agonists or antagonists of thefactor-encoding gene. The antagonists may be used to decrease theactivity of the factors and thus may be useful in the therapy ofdiseases associated with overactivity of a transcriptional regulatoryfactor. Such agonists or antagonists identified by assays employing thefactor-encoding genes of this invention are within the scope of thisinvention.

The present invention is useful as a means of controlling activation ina host cell of an NF-kB precursor, which results in formation ofactivated NF-kB, which, in turn, plays a key role in transcriptionalactivation of other sequences, such as the k light chain enhancer, theHIV enhancer and the interleukin-2 receptor α-chain gene. NF-kB has beenshown to be a ubiquitous inducible transcription factor; it has beenshown, as described herein, to be present in many types of cells (i.e.,all cell types assessed to date). It serves to make immediate earlyresponses which it is capable of effecting because it ispost-translationally activated. As a result, the method and compositionof the present invention can be used to control transcriptionalactivation of genes encoding a selected cellular protein. Changes inexpression of genes transcribed by RNA polymerase II in response toagents, such as steroid hormones, growth factors, interferon, tumorpromoters, heavy metal ions and heat shock are mediated throughcis-acting DNA sequence elements such as enhancers. Binding of NF-kBtranscription factor has been shown to confer transcriptional activityon several genes. Expression of these genes and others similarlyaffected can be controlled by the present method. For example, it hasbeen shown that expression of one of the two elements of the cellsurface receptor specific for IL-2 is controlled by NF-kB. Thus, in Tcells, which produce IL-2, production can be controlled (enhanced,reduced) by controlling activation of NF-kB. In a similar manner, themethod of the present invention can be used to control expression ofhuman immunodeficiency virus in infected host cells.

Methods and compositions of the present invention are based on use ofthe role of NF-κB as a second messenger, or mediator, in the expressionof genes in a wide variety of cell types. The expression of a genehaving an NF-κB binding recognition sequence can be positively ornegatively regulated to provide, respectively, for increased ordecreased production of the protein whose expression is mediated byNF-κB. Furthermore, genes which do not, in their wild type form, haveNF-κB recognition sequences can be placed under the control of NF-κB byinserting NF-κB binding site in an appropriate position, usingtechniques known to those skilled in the art,

DNA sequences known to contain NF-κB binding domains are shown in Table2. According to the methods described herein, the expression of genesunder the control of one of these elements is subject to modulation byalteration of the concentration or availability of NF-κB. This can alsobe carried out, according to the present method, for any gene whichcontains an NF-κB binding site. Furthermore, genes which do notnaturally contain NF-κB binding sites can be modified, using knowntechniques, to subject these genes to NF-κB modulation. First, anappropriate expression vector is selected for use in a biological systemof interest, the vector having a gene of interest and restriction enzymerecognition sequences to facilitate the insertion of a DNA fragmentcarrying the NF-κB binding sites.

For example, the sequences of the κ immunoglobulin enhancer, the SV40 70base pair repeat, the HIV long terminal repeat, the MHC class I H2-kbgene and the interferon β PRDII gene, all possess NF-κB binding sites(Table 2). By comparing sequences to which NF-κB binds specifically, aconsensus sequence has been determined: ##STR1## DNA sequences whichflank the binding site are scanned for convenient restriction enzymerecognition sequences to facilitate removal of the fragment from thelonger sequence in which occurs and its subsequent insertion into theexpression vector. If such sequences are present, the transfer of thefragment carrying the binding site, to the expression vector, isstraight forward. If convenient sites do not exist, fragment transfer isfacilitated through the introduction of such restriction enzymerecognition sequences using well known, site-directed mutagenictechniques. The construct, prepared as described, can then be introducedinto a biological system of interest.

The expression of such constructs in a biological system is subject tomodulation by NF-κB. For example, purified NF-κB could be introducedinto the system in an effective amount such that any inhibitory moleculepresent in the system would be titrated out and uninhibited NF-κB couldinteract with its binding recognition sequence, thereby increasing therate of transcription. This is an example of positive regulation.

Similarly, a copy of the NF-κB gene, cloned in an appropriate expressionvector, could be introduced into the biological system, therebyproviding for internal expression of the NF-κB molecule, preferably atrelatively high levels. Again, high levels of NF-κB would function totitrate out any inhibitor molecule present, and also to increase therate of transcription from a gene possessing a NF-κB binding site.

A level of discrimination among members of a related family of NF-κBbinding sites, by a modified NF-κB molecule, can also be introduced.Referring to Table 1, for example, there are apparent differences amongthe various NF-κB binding sites from various genes. A copy of a clonedNF-κB gene can be mutagenized to alter the binding domain by well knowntechniques, such as site or region directed mutagenesis. Alternatively,DNA fragments encoding a modified NF-κB binding domain can be madesynthetically, having appropriate cohesive or blunt termini tofacilitate insertion into the NF-κB gene to replace the existingsequences encoding the corresponding portion of the binding domain. Suchrestriction fragments can be synthesized having any desired nucleotidechanges. Mutated proteins encoded by such genes can be expressed andassayed for preferential binding to, for example, one of the 10different DNA binding sites shown in Table 1 or related members of thefamily of NF-κB binding sites. An example of an assay which can be usedto screen large numbers of recombinant clones in order to identifybinding domain mutants is that described by Singh et al., (Cell,52:415-423 (1988)). Once such a mutant is identified, a DNA expressionvector encoding this mutant protein can be introduced into a cell. Themutant protein will preferentially bind to the selected member ormembers of the family of DNA binding sites, such as those shown in Table2, thereby preferentially enhancing transcription from only those geneswhich contain that particular binding site.

                  TABLE 2                                                         ______________________________________                                        Sequences recognized by NF-κB.                                          Gene                 Sequence                                                 ______________________________________                                        Ig κ enhancer - mouse                                                                        GGGGACTTTCC                                              SV40 enhancer                                                                 HIV-1 (-91)                                                                   CMV (4).sup.1,2                                                               HIV-1 (-105)         AGGGACTTTCC                                              HIV-2                                                                         CMV (1).sup.1                                                                 β2-microglobulin                                                         serum amyloid A - g.sup.9                                                     Ig κ enhancer - human                                                                        GGGGATTTCC                                               CMV (3).sup.1                                                                 Interferon-β -- PRDII                                                                         GGGAAATTCC                                               CMV (2).sup.1        GGGACTTTCC                                               MHC class II - E.sub.α.sup.d                                                                 GGGACTTCCC                                               IL-2 lymphokine      GGGATTTCAC                                               mouse IL-2Rα   GGGGATTCCT                                               human IL-2Rα   GGGAATCTCC                                               MHC class I - H2 - K.sup.b                                                                         GGGATTCCCC                                               HLA - A2, A11, B7                                                             B27, B51                                                                      ______________________________________                                        CONSENSUS.sup.3 :                                                                                   ##STR2##                                                ______________________________________                                         .sup.1 In this particular element, the sequence has not been tested in a      binding assay. All others have been proven by direct binding and usually      by inhibition of binding to the Ig κ sequence.                          .sup.2 Since there are four putative NFKB recognition sites in the            cytomegalovirus enhancer, these have been numbered 1-4 as they are found      from 5' to 3' on the coding strand.                                           .sup.3 Consensus is based on all sequences through the assignments of the     sixth and tenth positions ingmore one deviant.                           

Negative regulation can be effected in an analogous manner. For example,a specific inhibitor molecule which is able to block (reduce oreliminate) NF-κB binding can be added to the biological system in aneffective amount. Preferably, this inhibitor is specific for NF-κB anddoes not interact with other cell constituents. An example of such amolecule is I-κB.

Alternatively, negative regulation can be effected using "decoy"molecules, which are designed to mimic a region of the gene whoseexpression would normally be induced by NF-KB. In this case, NF-κB wouldbind the decoy and, thus, not be available to bind its natural target.

Furthermore, in the case of an inhibitor molecule which is also aprotein, the gene encoding the inhibitor molecule can be identified,isolated, and cloned into an appropriate expression vector using commonmethodology. When introduced into an appropriate biological system, theinhibitor molecule is synthesized and functions to interact with NF-κBwith its binding site and as a consequence reducing the level oftranscription of the gene containing the NF-κB binding site.

Yet another method for negatively regulating the expression of a genecontaining an NF-κB binding domain involves the introduction of aneffective amount of a decoy sequence encoding the NF-κB binding domain.The decoy sequence serves as an unproductive binding domain with whichthe NF-κB molecule binds. As the finite number of NF-κB molecules bindto the decoy sequences, the number which bind productively (result inincreased transcription) with an intact gene, decreases.

Negative regulation can also be effected by the introduction of"dominantly interfering" molecules (see e.g., Friedman et al., Nature,335:452-454 (1988). For example, if the DNA binding domain and the DNApolymerase activating domain of NF-κB are spatially distinct in themolecule, a truncated form of the NF-κB molecule can be synthesized,using well known techniques. A preferred embodiment would be a truncatedmolecule retaining the DNA binding domain, but lacking the RNApolymerase activating domain. Such a "dominantly interfering" moleculewould recognize and bind to the NF-κB binding site, however, the bindingwould be non-productive. Because the activation portion of NF-κB wouldbe required for enhanced transcription, the truncated molecule wouldexert no positive effect. Furthermore, its occupation of the NF-κBbinding site effectively blocks access to any intact NF-κB moleculewhich may be present in the cell.

The invention is further illustrated by the following examples, whichare not intended to be limiting in any way.

EXAMPLES EXAMPLE 1

Identification of Nuclear Factor IgNF-A

METHODS

1. Gel electrophoresis DNA Binding assays with the SfaNI-SfaNI κpromoter fragment.

The SfaNI fragment was subcdoned into the SmaI site of pS64(pSPIgV.sub.κ, provided by N. Speck). For binding analysis this fragmentwas excised from pSPIgV.sub.κ by digesting with Hind III and Eco RI.These latter sites flank the Sma I site in the polylinker of pSP64.After end-labeling with α-³² P!dATP and the large fragment of E. coliDNA polymerase I, the radiolabeled fragment was isolated bypolyacrylamide gel electrophoresis. Binding reactions were performed andthe reaction mixtures resolved by electrophoresis. The ³² Plabeledfragment (about 0.5 ng, 10,000 cpm) was incubated with a nuclear extractof a human B lymphoma cell line (EW)(prepared by the method of Dignam,J. D. et al. Nucl. Acids Res. 11 1475-1489 (1983)) in the absence orpresence of two different non-specific competitor DNAs. Bindingreactions (25 μl) contained 10 mM Tris.HCl (pH 7.5), 50 mM NaCl, 1 mMDTT, 1 mM EDTA, 5% glycerol and 8 μg EW nuclear extract protein.Reactions 2-6 additionally contained 800, 1600, 2400, 3200 and 4000 ηg,respectively, of poly(dI-dC)-poly(dI-dC). Reactions 7-11 contained 300,600, 900, 1200 and 1500 ηg, respectively, of Hind I digested E. colichromosomal DNA. After a 30 min incubation at room temperature, theresulting complexes were resolved in a low ionic strength 4%polyacrylamide gel (acrylamide:bisacrylamide weight ratio of 30:1)containing 6.7 mM Tris.HCl (pH 7.5), 3.3 mM Na-acetate and 1 mM Na-EDTA.See Strauss, F. and Varshavsky, A. Cell 37 889-901 (1984). The gel waspreelectrophoresed for 30 min at 11V/cm. Electrophoresis was carried outat the same voltage gradient for 90 min at room temperature with bufferrecirculation. The gel was then dried and auto- radiographed at -70° C.with a screen.

Binding assays were performed as detailed above using 2400 ngpoly(dI-dC)-poly(dI-dC)) and the following DNA fragments: κ SfaNI-SfaNI(˜0.5 ηg, 10,000 cpm), κ PvuII-KpnI (˜0.5 ηg, 10,000 cpm), η PvuII-SfaNI(˜0.1 ηg, 5000 cpm) and pSP64 PvuII-EcoRI (˜0.2 ng, 5000 cpm). The κPvuII-SfaNI fragment was derived from the plasmid pSPIgV.sub.κ bydigesting with PvuII and EcoRI. The EcoRI site is in the polylinker andtherefore this fragment contains 16 bp of polylinker sequence.

2. Binding competition analysis in nuclear extracts of human EW and HeLacells

EW nuclear extract. Binding assays were performed as detailed aboveusing radiolabeled K PvuII-SfaNI fragment (˜0.1 ηg, 5000 cpm) and 2400ηg poly(dI-dC)-poly(dI-dC). Reactions 2-4 additionally contained 50, 100and 200 ηg, respectively, of the bacterial plasmid pSP64 whereas 5-7contained 50, 100 and 200 ηg, respectively, of the recombinant plasmidpSPIgV.sub.κ. Assuming that a molecule of pSPIgV.sub.κ contains a singlehigh affinity site whereas a molecule of pSP64 (3000 bp) contains 6000non-specific sites, the apparent affinity ratio of the factor for thesetwo types of sites is greater than 6000×200/50=12.4×10⁴ Hela nuclearextract. Binding assays were performed as detailed above usingradiolabeled κ PvuII-SfaNI fragment (about 0.1 ng, 5000 cpm), 2400 ngpoly(dI-dC)-poly(dI-dC) and 6 μg Hela nuclear extract protein (providedby P. Grabowski). Reactions 1 and 2 additionally contained 100 ηg ofpSP64 and pSPIgV.sub.κ, respectively.

3. DNase footprinting of factor-DNA complexes

The B cell nuclear extract was applied to a heparin-sepharose columnequilibrated with 10 mM Hepes pH 7.9, 20% glycerol, 1 mM DTT, 1 mM EDTA,5 mM MgCl₂ and 0.1M KCl. The κ-promoter binding factor was eluted with a0.25M KCl step. Binding reactions with this fraction (30 μl) contained2.5 mM MgCl₂, κ PvuII-SfaNI (˜1 ηg, 100,000 cpm) and 4800 ηgpoly(dI-dC)-poly(dI-dC) in addition to components detailed above. Thecoding strand of the κ promoter probe was 3' end-labeled (Eco RI site)with α-³² P! dATP using the large fragment of E. coli DNA polymerase.Reaction 1 was digested with DNase I (5 μg/ml) for 2.5 min at roomtemperature in the absence of B cell nuclear protein. Reaction 2 wasinitially incubated with the heparin Sepharose fraction of the EWnuclear factor (14 μg protein) for 15 min at room temperature and thendigested with DNase I as above. Each reaction was stopped with EDTA (5mM) and the products separated by native polyacrylamide gelelectrophoresis as detailed above. After autoradiography to visualizethe various species, DNA was eluted from the free (reaction 1) and bound(B1 and B2, reaction 2) fragment bands by incubating gel slices in 0.5Mammonium acetate (ph 7.5), 0.1% SDS and 1 mM EDTA with shaking at 37° C.overnight. The supernatants were extracted sequentially withphenol-chloroformisoamylalcohol (25:24:1 v/v) andchloroform-isoamylalcohol (24:1 v/v) and precipitated with 2 volumes ofethanol in the presence of carrier tRNA. After a reprecipitation stepthe products were analyzed by separation in a 10% polyacrylamide gel(20:1) in the presence of 8M urea followed by autoradiography at -70° C.with a screen. Lane 1 contained products of free fragment digestion fromreaction 1. Lanes 2 and 3 contained digestion products eluted from boundbands B1 and B2, respectively, from reaction 1. Lanes 1', 2' and 3'correspond to 1, 2 and 3, respectively, with the exception that theformer set was digested with DNase 1 for 5 min. A-G chemical cleavageladders of the κ promoter probe were coelectrophoresed to map thebinding domain. See Maxam, A. and Gilbert, W. Meth. Enzymol. 65, 499-525(1980).

4. Binding of a common nuclear factor to three Ig transcriptionalcontrol elements

Nucleotide sequences of actual and putative binding sites, FIG. 2. TheV_(L) binding site is defined by the DNase I protection assay (*indicates boundaries of the protected region). The V_(H) and J_(H)-C_(U) sequences are putative binding sites in the V₁₇.2.25 promoter andthe mouse heavy chain enhancer, respectively. Numbers in bracketsindicate start coordinated of octamer motif. Binding competitions.Binding assays (10 μl) were performed as detailed above using 1600 ngpoly(dI-dC)-poly(dI-dC) and the heparin Sepharose fraction of the EWnuclear factor (1.5 μg protein). V_(L) probe (about 0.1 ηg, 5000 cpm)lanes 1-3, 10, 11. V_(H) probe (˜0.2 ηg, 5000 cpm) lanes 4-6, 12-12.J_(H) -C.sub.μ probe (˜0.2 ηg, 5000 cpm), lanes 7-9, 14, 15. Lanes 2, 5,8 additionally contained 5 ng of a V_(L) promoter oligomer (36 bp,spanning positions -81 to -44 of the MOPC-41 V.sub.κ gene segment)whereas lanes 3, 6, 9 contained 50 ng of the same oligomer. Lanes 11,13, 15 additionally contained 50 ηg of a J_(H) -C_(H) oligomer (41 bp,spanning positions -1 to 40 of the heavy chain enhancer). Complementarysingle-stranded synthetic oligonucleotides were kindly made by Dr.Ronald Mertz, Genzentrum der Universitat Munchen and Dr. E. L.Winnacker, Institut fur Biochemie der Universitat Munchen. They wereannealed prior to use as competition substrates in the binding assay.

RESULTS

A radiolabeled SfaNI-SfaNI DNA fragment derived from the upstream regionof the MOPC 41 κ light chain gene (FIG. 1) was incubated with a nuclearextract of a human B cell line in the absence or presence of twodifferent competitor DNAs. The resulting complexes were resolved fromthe free fragment by electrophoresis through a low ionic strength,non-denaturing polyacrylamide gel and visualized by autoradiography. Inthe absence of competitor DNA all of the labeled fragment was retainedat the top of the gel probably due to the binding of an excess of nonsequence-specific proteins. With addition of increasing amounts ofeither poly(dI-dC)-poly(dI-dC) or E. coli chromosomal DNA ascompetitors, putative protein-DNA complexes which migrated more slowlythan the free fragment were detected. The relative abundance of themajor species of complex (B) as well as that of minor species wassignificantly greater in the presence of the alternating copolymercompetitor DNA. Because substitution of the simple copolymerconsiderably increased the sensitivity of the assay, it was used in allsubsequent binding analyses.

To test the sequence-specificity of the major species detected in thebinding assay, a set of mutually overlapping Kappa promoter fragments(See FIG. 1, SfaNI-SfaNI, PvuII-KpnI and PvuII-SfaNI) and a similarlength fragment derived from the bacterial plasmid SP64 (EcoRI-PvuII)were individually assayed. Whereas the bacterial DNA fragment showed noappreciable binding all of the κ promoter fragments yielded majordiscrete complexes of similar mobilities. The mobilities of a series ofcomplexes formed with different length fragment probes (75-300 bp) areapproximately the same (data not shown). These data therefore suggestedthe binding of a specific nuclear factor within the region of overlap ofthe Kappa promoter fragments. This region includes the conservedoctameric sequence but not the TATA element. Note that with the smallest75 bp Kappa promoter fragment no appreciable label was retained at thetop of the gel. Thus, as has been noted recently, the use of small probefragments further enhances the sensitivity of detection of specificprotein-DNA complexes.

The sensitivity gained by use of both the copolymer and a small fragmentprobe permitted the detection of two complexes, B1 and B2. The majorspecies B1 corresponds to complex B in the earlier figure. The relativeaffinity of the factor(s) for κ promoter DNA was estimated by acompetition assay. Whereas a control plasmid (pSP64), when added in theabove incubation, failed to compete for binding in the concentrationrange tested, the recombinant plasmid (pSPIgV.sub.κ) effectively reducedthe formation of both species B1 and B2 in the same range. The latterplasmid was constructed by insertion of the upstream region of the Kappapromoter into pSP64. Assuming that the pSPIgV.sub.κ plasmid contains asingle high affinity binding site these results suggest that the nuclearfactor(s) responsible for B₁ and B2 has at least a 10⁴ -fold higheraffinity for its cognate sequence than for heterologous plasmid DNA (seeMethods section 2 above).

To determine if the factor(s) responsible for formation of B1 and B2 wasspecific to B lymphocytes, a nuclear extract derived from human HeLacells was assayed for binding to the κ promoter probe. Both species B1and B2 were generated at similar levels as that observed with B cellextracts, by the HeLa extract. Furthermore, both were specificallycompeted by the pSPIgV.sub.κ plasmid. Thus HeLa cells also contain afactor(s) which binds specifically to the κ promoter upstream region.

DNase I footprint analysis was used to delineate at a higher resolutionthe binding domain(s) of factor(s) present in complexes B1 and B2. Tofacilitate these studies, the binding factor(s) from B cells waspartially purified by chromatography of nuclear extract protein on aheparin sepharose column. Most of the binding activity eluted in a 0.25MKCl step fraction, giving a purification of approximately 5-fold. Forfootprint analysis, DNase I was added for a partial digestion afterincubation of the partially purified factor(s) with the κ promoter probeB1 and B2 species were then resolved from free fragment bypolyacrylamide gel electrophoresis. Bound DNA was eluted from both B1and B2 bands and examined by denaturing polyacrylamide gelelectro-phoresis. DNase I digests of the e promoter probe in the absenceof B cell protein and A+C chemical cleavage ladders werecoelectrophoresed to map the binding domain. Factor(s) in the B1complexes appeared to protect a 19 nucleotide region on the codingstrand. The 5' and 3' boundaries of the protected region map topositions -72 and -52, respectively, from the site of transcriptionalinitiation. The region of DNase I protection was centered about theconserved octanucleotide sequence ATTTGCAT suggesting its importance inthe recognition of the Ig promoter by the nuclear factor. B2 complexesshowed a virtually identical DNase I protection pattern as B1 complexesand therefore do not appear to involve additional DNA contacts. Thesimplest interpretation of this observation is that the B2 complex isgenerated by dimerization through protein-protein interactions of thefactor responsible for the B1 complex. Alternatively, the B2 complexcould be formed either by the binding of another protein to the factorresponsible for the B1 complex or by recognition of the same set ofsequences by a distinct DNA binding protein.

Because the octamer sequence motif is present in both light and heavychain gene promoters as well as in the enhancer elements of both mouseand human heavy chain genes, assays were performed for factor binding tofragments from a mouse heavy chain promoter (V_(H)) and the mouse heavychain enhancer. The V_(H) promoter fragment was derived from the 5'region of the V₁₇.2.25 gene and included nucleotides between positions-154 and +57 relative to the transcriptional start site. Grosscheal, R.and Baltimore, D. Cell 41 885-897 (1985). In this promoter the conservedoctanucleotide spans positions -57 to -50 (FIG. 9). The heavy chainenhancer fragment was derived from the germline J_(H) C.sub.μ region andspanned positions 81 to 251 within a 313 bp region implicated inenhancer function. Banerji, T. et al. Cell 33 729-740 (1983). Theconserved octanucleotide is positioned between coordinates 166 to 173 inthe above fragment (FIG. 2). The B-cell heparin sepharose fraction(purified on the basis of binding to the Kappa promoter sequence)evidenced binding to both the V_(H) promoter fragment and to theenhancer fragment. The mobilities of the complexes formed with the threefragments were very similar consistent with the binding of a commonfactor. Furthermore, binding to these fragments was specificallycompeted by a synthetic duplex 40-mer that spanned the octanucleotidemotif of the MOPC 41 κ light chain gene promoter. An oligomer ofequivalent size containing a sequence from a region of the mouse heavychain enhancer lacking the octanucleotide motif failed to compete forbinding in the same concentration range. This competition analysisfurther strengthens the suggestion that a common nuclear factor (IgNF-A)binds to all three Ig transcriptional elements. As has been mentionedpreviously, these three transcriptional elements share an identicalsequence motif ATTTGCAT (FIG. 2). Thus, the binding of a common nuclearfactor is almost certainly mediated by this motif.

EXAMPLE 2

Dependency of in vitro transcription of Ig genes on an upstream sequence

METHODS

FIG. 3: Templates. The deletions 5'Δ5 and 5'Δ7 have been describedbefore. See Bergmah Y. et al PNAS U.S.A. 81:7041 (1981). The highlyconserved octanucleotide sequence which is found upstream of allsequenced heavy and light chain variable region genes is boxed (labelled"OCTA"). It is located approximately 30 base pairs upstream from the"TATA" box. The plasmids pκ and pΔκ were constructed by converting the5'-ends of 5'Δ5 and 5'Δ7 into a Hind III site by means of syntheticlinkers followed by cloning the fragment up to the Bgl II site in theJ.sub.κ -C.sub.κ major intron into Hind III, Bam HI digested pUC-13.pκE.sub.κ and pκE.sub.κ represent plasmids containing either the kappaenhancer of the heavy chain enhaner cloned into the unique Hind III siteof pK. The segments used as the enhancers are an 800 bp Hind III-Mbo IIfragment from the J.sub.κ -C.sub.κ intron (Max, E. E. et al. (1981) J.Biol. Chem. 2565116) and a 700 bp Xba I-Eco RI fragment from the J_(H)-C.sub.μ intron. Gillies, S. D. et al. Cell 33:717 (1983); Banjerji, J.et al. Cell 33:729 (1983).

The cell lines RAMOS and EW were grown in RPMI medium containing 10%inactivated fetal calf serum to a density of 5-8×10⁵ cells per ml. Wholecell extracts were generated according to the procedure of Manley etal., PNAS U.S.A. 77:3855 (1980), and had a final protein concentrationof approximately 18 mg/ml. Run off transcription reactions were carriedout at 30° for 60' in a reaction volume of 20 μl. A typical reaction mixcontained 9 ul (160 μg) of whole cell extract, 50 uM each of ATP, CTPand GTP, 0.5 uM UTP, 10 μCi of α-³² P UTP (NEG 007×, 7600 Ci/mM) 5 mMcreatine phosphate, 0.3 mg/ml creatine phosphokinase (Sigma), 12 mMHepes 7.9, 12% glycerol, 60 mM KCl, 5 mM MG⁺⁺, 1 mM EDTA, 0.6 mM DTT,linearized template (about 50 ηg) and poly (dI-dC)-poly(dI-dc) as anon-specific carrier (about 400 ηg). The reactions were terminated byadding 200 μl of stop buffer (7M urea, 100 mM LiCl, 0.5% SDS, 10 mMEDTA, 250 μg/ml tRNA, 10 mM Tris (pH 7.9), followed by two extractionswith phenol: chloroform: isoamyl alcohol (1:1:0.05), one with chloroformand precipitation with ethanol. The RNA's were treated with glyoxal andanalyzed by electrophoresis through a 1.4% agarose gel in 10 mM sodiumphosphate (pH 6.8), 1 mM EDTA. See Manley et al. supra. The gel was thendried for autoradiography with an intensifying screen at -70° C. Run offtranscripts were obtained utilizing templates containing either the wildtype promoter or the truncated Kappa promoter. In vitro transcriptionused closed circular templates containing the wild type promoter or thetruncated κ promoter. In these reactions 50 ng of a closed circulartemplate containing the adenovirus major late promoter (MLP) wasincluded as an internal control. The transcripts specific to the κtemplate or the adenovirus template are indicated as κ and MLP,respectively. For a template containing the major late promoter theplasmid pFLBH was used. The plasmid contains sequences from 14.7 to 17.0map units of adenovirus inserted between the Bam HI and Hind III sitesof pBR322 and was the kind gift of A. Fire and M. Samuels.

Either the linearized or the supercoiled template (50 ηg) was incubatedin a volume of 20 μl with 9 μl (about 150 μg) of EW extract, 6% (wt/vol)polyethylene glycerol 20,000 and all other components described exceptthe nucleotides for a period of 60 minutes at 30° C. Transcription wasinitiated by the addition of nucleotides and radioactive UTP to thefollowing final concentrations: 60 uM each of ATP, CTP and GTP and 1 μMUTP and 10 μCi α-³² P UTP (NEG 007×, 600 Ci/mM). The initiating pulsewas maintained for 10' at 30° followed by a 10' chase with a vast excessof non-radioactive nucleotides. Final concentrations during the chasewere as follows: 330 μM ATP, CTP, GTP and 1 mM UTP. The reactions werequenched, worked up and the run off transcripts analyzed as describedabove. Mapping of the initiation site of the transcript was conducted asfollows: Transcripts generated from closed circular templates were takenup in 20 μl of HE (50 mM Hepes, pH 0.7, 1 mM EDTA) and 10 μl was usedfor hybridization selection. A hybridization template complementary tothe κ RNA was constructed by cloning the Pva II-Sau 3A fragment whichcontains the cap site of the MOPC41 gene (Queen and Baltimore, Cell33:741 (1983)) into the M13 phage MP9. Single stranded phage DNA wasprepared and purified by densitycesium chlcentrifugation through cesiumchloride. MLP specific transcripts were detected using the M13 cloneXH11 provided by A. Fire and M. Samuels. Hybridizations were done in afinal volume of 15 μl in the presence of 750 mM NaCl and 100-200 ug ofsingle stranded complementary DNA at 50° C. for 2 hrs. The reactionswere then diluted with 200 μl of cold quench solution (0.2M NaCl, 10 mMHepes pH 7.5, 1 mM EDTA) and 2 U of ribonuclease T1 added. Digestion ofsingle stranded RNA was allowed to proceed for 30' at 30° C. after whichthe reactions were extracted once with phenol chloroform isoamyl alcohol(1:1:0.05) and precipitated with carrier tRNA. The pellet was washedonce with cold 70% ETOH, dried and resuspended in 80% v/v formamide, 50mM Tris borate, pH 8.3 and 1 mM EDTA. The RNA was denatured at 95° C.for 3 min and then electrophoresed through a 6% polyacrylamide 8.3M ureasequencing gel. The upper of 2 bands (κ) derived from the immunoglobulinpromoter represent the correct start for κ transcription. The lower bandis seen at variable intensities and probably does not represent adifferent cap site, as explained below.

RESULTS

Whole cell extracts were made from two human Burkitt lymphoma lines, EWand RAMOS, by the procedure of Manley et al. supra. The templates usedfor in vitro transcription reactions are diagrammed in FIG. 3. Thetemplate representing the wild type gene (pK) was derived from theMOPC41 κ gene and contained sequences from approximately 100 bp upstreamfrom the transcription initiation site (end point 5'Δ5, FIG. 3) to theBgl II site in the major J.sub.κ -C.sub.κ intron Max, E. E. J. Biol.Chem. 256:5116. This fragment retains the complete variable region whichis rearranged to J.sub.κ l, but not the κ enhancer which is furtherdownstream of the Bgl II site. See, e.g., Queen and Stafford, Mol. Cell.Biol. 4:1042 (1984). This short 5' flank has been shown to be sufficientfor accurate initiation and high level of transcription in a transienttransfection assay. Bergman, Y. et al. PNAS U.S.A. 81:7041 (1984).Deletion analysis of the κ promoter showed previously that importantregulatory sequences are present between nucleotides -79 and -44 becausedeletion 5'Δ7 completely abolished transcriptional competence of thegene while deletion 5'Δ5 had no effect. See Bergman et al. supra. Thetemplate representing an inactive promoter mutant (pΔκ) was constructedby engineering a Hind III site into the 5'-end of 5'Δ7 and cloning thesegment of the gene up to the Bgl II site into pUC13 cut with Hind IIIand Bam HI.

To examine transcriptional activity in B cell extracts, a lineartemplate truncated at the Sac I site in the polylinker was used andtranscripts ending at this site (run off transcripts) were examined byelectrophoretic separation. A run off transcript of 2.3 kb was evidentwhen RAMOS, EW or HeLa cell extracts were used. When a κ chain enhancersequence was added to the construct, no effect was evident implying thattranscription in these extracts is enhancer independent. (In EW andHeLa, the enhancer appears to cause a slight increase in the backgroundradioactivity but not in the 2.3 kb band.) The band at 2.3 kb could beabolished by not adding the template or by transcribing in the presenceof 0.5 μg/ml amanitin. Thus it represents a template-specific, RNApolymerase II transcript. The band just below 2.3 kb is not decreased byα-amanitin and presumably reflects end-labeling of endogenous 18S rRNA.

To assess whether initiation of transcription occurred at the naturalcap site, a second assay was used. See Hansen, U. and P. A. Sharp (1983)EMBOJ 2:2293. For this assay, the uniformly labeled RNA was hybridizedto a single stranded DNA probe spanning the transcription initiationsite (generated by cloning the Pvu II-Sau 3A fragment of the κ gene intophage M13). The resulting complex was digested with ribonuclease Tl andthe ribo-nuclease-resistant RNA fragments were analyzed byelectrophoresis through a 6% polyacrylamide gel with 8.3M urea. Theupper band (labeled κ) represents the correct cap site. The band justbelow it was seen at variable intensities and probably does notrepresent a different cap site but rather arises from cleavage withribonuclease T1 at the next G residue from the 3'-end of the protectedregion. (Examination of the sequence near the Sau 3A1 site shows thatthe second set of G residues on the RNA lies 19 bp upstream from the endof the region of homology with the single stranded DNA probe). Thus theextracts generated from B cells were capable of correctly initiating andtranscribing the immunoglobulin promoter in vitro with approximately thesame efficiency as a HeLa cell extract.

To analyze the effect of 5' flanking sequences in vitro, we examined thetranscription of the deleted gene, pΔK. Because many regulatory effectsact upon the rate of initiation of transcription, we chose to use apreincubation, pulse-chase protocol which measures the initiation rates.See Fire, A. et al. J. Biol. Chem. 259:2509 (1984). The template DNA wasfirst preincubated with the extract to form a pre-initiation complex.Transcription was then initiated by the addition of nucleotides andradiolabeled UTP. The initiated transcripts were completed during achase period with unlabelled nucleotides and analyzed by electrophoreticseparation. Incorporated radioactivity in this assay is proportional tothe number of correct initiations occurring during the pulse.

The template pκ, which contains about 100 bp upstream of the initiationsite, initiated approximately 10-fold more efficiently than did thedeletion mutant, pκ. Again, the presence of the heavy chain enhancerplaced at -44 bp to the truncated promoter did not alter the level oftranscription. When closed circular templates were used, a similareffect of the promoter truncation was observed. In these reactions atemplate containing the major late promoter of adenovirus was includedas an internal control; the expected protected RNA fragment of 180 bp islabeled MLP. There was a 10-fold decrease in the efficiency oftranscription from the mutant promoter, whereas the transcript of themajor late promoter remained constant. The reason for the apparentdecrease in the amount of transcription from the supercoiled templatecontaining the heavy chain enhancer has not been further addressed. Itis evident, however, that the dependence of transcription on an upstreamsequence between -44 and -79 is observed whether the effect describedabove was specific to B cell extracts, the same templates weretranscribed in the heterologous HeLa whole cell extract. A 4- to 5-folddecrease in transcription was seen with the deleted template whencompared with the wild type template. Thus, the effect of the deletionis at best, only modestly tissue-specific.

We have reported here the development of transcriptionally competentwhole cell and nuclear extracts from two independent human B celllymphomas. In such extracts, transcription from the promoter of theMOPC41 κ gene was correctly initiated and a promoter deletionsignificantly reduced the level of initiated RNA. In vitro, the effectof the deletion used here is several hundredfold when analyzed by atransient transfection assay. However, the effect observed in vitro isonly about 10- to 15-fold. Although there are now several examples ofupstream sequence requirements for in vitro transcriptions, See, e.g.,Groschedl R. and Birsteil M. L. (1982) PNAS U.S.A. 79:297; Hen, R. etal. (1982) PNAS U.S.A. 79:7132, the effects have been smaller than thecorresponding one in vivo. This is possibly due to the dominance of theTATA box and associated factors in determining the level oftranscription in vitro Miyamoto, N. G. et al., Nucl. Acids Res. 12:8779(1984).

EXAMPLE 3

Discovery and Characterization of IgNF-B

The mobility shift gel electrophoresis assay was used to screen nuclearextracts from a variety of cell lines for octamer binding proteins. Theband corresponding to IgNF-A was found in all extracts but a second bandwith distinct mobility from IgNF-A was found only in nuclear extractsfrom lymphoid cells. This lymphoid-specific octamer binding protein,termed IgNF-B, was found in nuclear extracts from all pre-B, mature Band myeloma cell lines tested and in nuclear extracts from some T celllyinphonas. IGNFB was not detected in nuclear extracts from thenon-lymphoid cell lines, Hela, ψ2, Cos and Mel. IgNF-B was shown to bespecific for the same octamer sequence as IgNF-A by competitionexperiments n which the IgNFB band was selectively competed byunlabelled DNA fragments sharing only the octamer sequence and not byDNA fragments lacking the octamer sequence.

The octamer sequence is found at approximately position -70 upstream ofthe transcription start site of all immunoglobulin (Ig) variable geneswhich is in the region that has been shown to control the lymphoidspecificity of the Ig promoter. Thus, IgNF-B binds to the upstreamoctamer sequence in lymphoid cells and activate transcription.

EXAMPLE 4

Factors Binding to μ-Enhancer: E Factor

The fully functional μ enhancer has been localized to a 700 bp XbaIEcoRI fragment from the major intron between J_(H) and C.sub.μ. Thisfragment can be further subdivided by cleaving at the PstI site togenerate a 400 bp Xbal-PstI fragment (μ400) and a 300 bp PstI-EcoRIfragment (μA300). It has been shown by transient transfections that30-50% of the tissue specific enhancer activity is retained in μ300,whereas there is no detectable activity of μ400. The gel binding assaywas employed to investigate what protein factors may interact with theu-enhancer. Briefly, end-labelled DNA fragments were incubated withnuclear extracts made from tissue culture cells. After 20 min at roomtemperature the mixture was loaded on a low ionic strengthpolyacrylamide gel and electrophoresis carried out at 120V for 2 hrs.The gels were then dried for autoradiography. When the functional 300 bp(μ300) enhancer fragment was used in such an assay a DNA-protein complexwas observed in extracts derived from the human B lymphoma cell line EW.To show that this new band represented a specific complex bindingreactions were carried out in the presence of varying amounts ofnon-radioactive competitor fragments. It is easily seen that when μ300is added as the competitor fragment, the complex band is completelylost. In contrast, the adjacent u400 fragment or a 450 bp fragmentcontaining the κ light chain enhancer, cause only a minor effect even atthe highest concentrations used. It is interesting to note that thereappears to be a slight increase in the amount of specific complex in thepresence of the κ enhancer fragment. As demonstrated below, both the μand the κ enhancers interact with at least one common protein and thisis not the factor being detected by binding the u300 fragment. Theincrease in the specific complex in the presence of the κ enhancer isprobably due to the removal of factors common to both the enhancers fromthe reaction mix, thus leaving more of the labelled fragment availableto bind to the μ specific factor being detected by it.

In order to be able to detect binding sites for less abundant proteinsand also to more precisely define the complex detected with μ300, thisfragment was further dissected. Each of the smaller fragments generatedwere analyzed for their ability to serve as binding sites for nuclearproteins. FIG. 5A shows a partial restriction map of the relevant regionof the μ enhancer. μ300 was digested with AluI, HinfI and DdeI togenerate a number of 50-70 bp fragments labelled μ50, μ(60)₂ (a mixtureof AluI-DdeI and HinfI to AluI) and μ70 (AluI-AluI). Binding reactionswere carried out with each of these fragments with nuclear extracts ofEW lymphoma cells in the presence of increasing amounts of thenon-specific competitor poly (dI-dC)-poly(dI-dC).

Fragment μ50 forms a major complex band that is barely decreased even inthe presence of 4 ug of poly(dI-dC)-poly(dI-dC). The mixture of the two60 bp fragments does not give rise to a discrete complex band. Finallythe μ70 fragment gave 2 faint, but discrete, nucleoprotein complex bandsof which the lower one is again barely affected by 3 ugm of non-specificcarrier poly(dI-C)-(dI-C).

Specificity of the complexes observed were shown by competitionexperiments using a variety of DNA fragments, FIG. 5B. Thus, the complexgenerated with μ50 is specifically competed away in the presence of μ300(of which μ50 is a part), or a κ promoter fragment, but not bycorresponding amounts of μ400 or a κ enhancer fragment, consistent withthe complex being generated by the interaction of the previouslydescribed factor IgNF-A with its cognate sequence. (This factorrecognizes a conserved octanucleotide, ATTTGCAT, found in the promotersof all sequenced immunoglobulin genes and within this subfragment of theheavy chain enhancer.) The complex observed with μ70 was specificallycompeted away by itself and to some extent with the κ enhancer but notat all by either the Moloney murine leukemia virus enhancer or by μ400.Further competition analysis showed that this complex could not becompeted away by either (μ60)₂, μ50 or μ70 (central AluI-AluI fragment).The binding we have observed is therefore specific to this smallfragment and was detected only upon further dissecting μ300 whichseparated the major observable interaction of IgNF-A with the enhancersequence to another fragment (μ50).

Ephrussi et al. and Church et al. have used methylation protectionexperiments to define a set of G residues within the heavy chainenhancer that are specifically resistant to methylation by DMS in Bcells. This result lead to the proposal that tissue-specific DNA bindingproteins were responsible for this decreased accessibility of thereagent to DNA. The protection was observed in 4 clusters, the DNAsequences of which were sufficiently homologous to derive a consensussequence for the binding site of a putative factor. All four postulatedbinding sites (E1-E4) are found within the 700 bp fragment; however μ300retains only 2 complete binding domains (E3 and E4) for this factor andthe octamer (0) sequence. The Alu-Alu fragment that shows that specificnucleoprotein complex described above contains the complete E3 domainand the factor we detect in vitro presumably is the same as thatdetected in vivo. Thus, it was unexpected that the HinfI-Dde fragment(μ50) containing E4 and 0 should not compete for binding to μ70.

In case this was due to the fragment predominantly binding IgNF-A at theoctamer site and thus making it unavailable as a competitor for μ70,binding reactions and competition assay were done for a fractiongenerated by chromatography of the crude extract over aheparin-sepharose column, that contained μ70 binding activity and wassignificantly depleted of IgNF-A. When μ50 or μ170 was endlabeled andincubated with the column fraction, no specific nucleoprotein complexeswere seen upon electrophoretic analysis. Even in this fraction, μ50 andμ170 failed to compete successfully for the interaction between μ⁷⁰ andits binding protein, thus implying strongly that the binding sitedefined as E4 perhaps does not bind the same factor that binds at E3.Similarly, the El domain (isolated as a Hinf-PstI fragment) does notcompete as effectively as μ70 itself for the binding of the factor toμ70.

To determine the location of the binding sites within individualfragments (μ70 and μ50), the technique of methylation interference wasemployed. End-labelled DNA fragments were partially methylated onguanines and adenines using dimethyl sulfate (DMS). Methylated DNA wasthen used for binding reactions with crude extracts and the complex wasresolved from the free fragment by electrophoresis. Both complex andfree fragment bands were then excised from the gel, and the DNA wasrecovered by electroelution. Piperidine cleavage of the recoveredfragments was followed by electrophoresis through 12% polyacylamide ureasequencing gels. In principle, if any of methyl groups introduced byreaction with DMS interfere with the binding of a specific protein thenthat molecule of DNA will be selectively missing in the complex formedand subsequently the corresponding ladder. The method therefore allowsidentification of G residues making intimate contacts with the protein.We have found that the use of DNaseI footprinting via the gel bindingassay to be complicated in the case of some of these less abundantfactors because of the short half lives of the complexes themselves.Thus, if a binding incubation is followed by partial DNaseI digestion,it is possible that in the course of time required to load the sampleand have the complex enter the gel, DNA fragments that were in complexform may exchange with the larger amounts of free fragment in thebinding reaction. Thus not leading to any distinction in the DNasepatterns seen with wither the complex or the free DNA (e.g. the halflife of the nucleoprotein complex in μ70 is less than 1 minute).

The result of carrying out such an interference experiment using nuclearextracts and on the u50 DNA fragment shows that the complex observedarises via interaction of the IgNF-A protein at the conserved octamericsequence. The free fragment generates a characteristic G ladder and thecomplex form is specifically depleted in DNA molecules carrying a methylgroup at the G residue indicated by the asterisk which lies in themiddle of the conserved octamer. Presumably, modification at thisresidue seriously impedes the formation of a stable complex between theprotein and its cognate sequence. This residue was also shown to beprotected against methylation of DMS in vivo. Interestingly, however,none of the other G residues observed to be protected in vivo in thisregion of the μ enhancers appear to be affected in our in vitrointerference experiment. Therefore, if these protections in vivo are dueto the binding of a protein, this factor is different from IgNF-A or Band is not binding to fragment in vitro.

On the μ70 fragment several G residues were identified as beingimportant in forming intimate contacts with the binding protein (E). Onthe coding strand bands the 3 G's are significantly reduced in intensityin the complex as compared to the free DNA fragment, and on thenon-coding strand 2, GCs are significantly affected.

The results of both the in vivo DMS protection experiment and the invitro methylation interference experiments are shown herein. The openand closed circles above the sequence were the residues identified byEphrussi et al. to be protected against methylation in vivo whereas theencircled G's are the ones identified by us in vitro. The pattern ofprotection and interference on the μ70 fragment over the consensussequence is strikingly similar in vivo and in vitro, which indicatedstrongly that the protein identified here by means of the gel bind assayis the one that interacts with this sequence in vivo. Analogous to μ50,however, the second set of protections seen in this region in vivo wasnot observed in vitro. Tissue specificity of the factors detected: Inorder to determined whether the proteins identified are limited toexpression only in B cells, a large number of extracts made from B cellsand non-B cells were screened. Complexes that co-migrate with the onesgenerated and characterized (by competition and methylation interferenceexperiments) in the B cell line EW, were observed on both the fragmentsμ50 and μ70 (μ70) in all the cell lines examined. Although the complexgenerated in each extract has not been further characterized, weinterpret this data as indicating that both these factors are non-tissuespecific. A second complex (NF-κB) was observed with the μ50 fragmentthat was restricted to B and T cells only.

A point to note is that although the amount of protein in each lane hasbeen held constant at between 9 and 11 μg, the extent of complexgenerated was found to vary considerably from extract to extract. Thus,showing that quantitive estimations of the abundance of proteins indifferent cell lines using this assay is not very meaningful at thisstage. (This is presumably due to subtle variations in the state of thecells and the extraction conditions for the different cell lines).

In summary, analysis of the functional 300 bp PstI-EcoRI fragment of thep enhancer reveals that:

(i) at least 2 different proteins bind within this DNA sequence. Oneprotein (IgNF-A) interacts with an octamer sequence (ATTTGCAT) that ishighly conserved upstream of all heavy and light chain variable regiongenes and is also found in the u enhancer. The second protein interactswith a sequence shown by Ephrussi and Church to be protected in a tissuespecific manner against methylation by DMS in whole cells;

(ii) both factors can be detected in nuclear extracts from a variety ofcell lines and are therefore not B-cell specific;

(iii) both E1 and E4 sequences hardly compete for the binding of thefactor to μ70 (which corresponds to E3), thus implying that thesesequences do not interact with the same factor, although the sequencehomology amongst the sites would have lead one to expect that theyshould.

EXAMPLE 5.

Identification of Factors binding to Kappa-light chain enhancer

An enhancer element has also been identified in the major intron of theκ light chain gene. Picard and Schaffner showed that the enhancementactivity can be localized to a ˜500 bp AluI-AluI fragment and Queen andStafford have further refined the 5' and 3' boundaries so that theenhancer may be considered restricted to 275 base pairs within thelarger fragment. We have dissected this region into a number of smallerfragments and assayed each of these by means of the gel binding assayfor the location of protein binding sites.

A restriction map of the relevant region of the κ enhancer is shown inFIG. 7. The black boxes represent sequences identified by Church et al.to be homologous to the putative protein binding domains detected in theμ enhancer in vivo.

Fragments generated by cutting with Dde and HaeIII (κ1, κ2, κ3, κ4 andκ5; FIG. 7) were assayed for binding in the presence of increasingamounts of poly(dI-dC)-(dI-dc) as a non-specific carrier, κ4 and κ5appeared to be obviously negative while κ3 and κ2 appeared to bepositive. Preliminary results show that the internal fragment does notcontain any specific binding sites either. The nucleoprotein complexbands generated with 0.5-1 ηg of radiolabelled probe could be detectedeven in the presence of 3 μg of the carrier.

To show that the bands detected represented a specific interactionbetween a protein and DNA, we carried out competition experiments. Thecompetition pattern for κ2 was strikingly similar to what had beenearlier observed with the μ70 fragment; relatively large amounts ofu400, the Moloney leukemia virus enhancer, the SV40 enhancer or the κpromoter (containing the conserved octa) to κ2 did not compete forbinding, although u300 and the κ enhancer did. Since κ2 contains aputative E box identified by sequence comparison (as does μ70) wecompeted its binding with smaller fragments from μ300. The complex isspecifically competed away by the addition of unlabelled μ70 during theincubation, but not by μ60, μ170 or the SV40 enhancer. Further, theprotein that binds to this sequence co-fractionates with the μ70 bindingactivity through two sequential chromatographic steps (Heparin agaroseand DEAE Sepharose). Thus, we conclude that the same sequence specificprotein binds to both the fragments μ70 and κ2 and that there is atleast one common protein interacting with both the μ and the κenhancers.

The κ3 complex failed to be competed away by μ300 (compare lanes 3 and 4with lane 2), μ400 as a κ promoter containing fragment. However, thecomplex was specifically competed away with both the complete κ enhancerand the SV40 enhancer. The band below the major κ3 complex was seen atvariable intensities in different experiments and failed to compete evenwith the complete κ enhancer in this experiment and has not been furtherinvestigated at this stage. The observation that the SV40 enhancerspecifically competes for binding of this factor is not altogethersurprising, since this fragment and the SV40 enhancer share an identicalstretch of 11 nucleotides.

The binding site of this factor on the κ3 fragment was localized bymethylation interference experiments. In two different extracts,methylation at three of a stretch of 4 residues within this sequencecompletely abolished binding. This stretch of G's forms a part of theconserved region (GGGGACTTTCC) between the SV40 enhancer and κ3. Thus,the binding site was localized towards one end of the κ3 fragment. Theresults also served to explain the specific competition observed earlierwith the SV40 Enhancer. Interestingly, deletion mapping of the κenhancer shows that sequences within the κ3 fragment are extremelyimportant for enhancer function.

The tissue range of this factor was examined by carrying out bindinganalysis with κ3 in extracts from a variety of cell lines. Nucleoproteincomplex formation κ3 was detected in a mouse B cell line, but not in 5other non-B cell lines. The ubiquitous factor detected by μ50 is presentin all these cell lines and served as a positive control for theexperiment. The factor κ3 therefore appears to be restricted toexpression to B lymphoid cells.

We then examined extracts made from cells at various stages of B celldifferentiation (FIG. 8). Interestingly κ3 binding protein can bedetected in the Abelson murine leukemia virus transformed pre-B cellline PD, in two mouse B cell lines (WEH1231 and AJ9, FIG. 8), one humanB cell line (EW, FIG. 8) and 2 human myelomas (KR12 and 8226, FIG. 8).However, it does not appear to be present in a pre-preB cell line (C5,FIG. 8) and in mouse pre-B cell lines (HATFL, 38B9, 70Z, FIG. 8). Thus,this factor appears to be not only tissue-specific, i.e., limited tocells of the B lymphoid lineage, but also stage-specific within thatlineage. In the series of extracts examined, the presence of this factorbears a striking correlation with κ expression.

The results with the Kappa enhancer can be summarized follows:Dissection of the κ enhancer enabled detection of two distinct bindingproteins with this DNA. One of these proteins appears to be ubiquitousand interacts with the u heavy chain enhancer as well. The secondprotein appears to be highly expressed in a stage-specific manner withinthe B cell lineage and can be detected only in those cell lines wherethe endogenous κ gene is active. There does not appear to be a bindingsite for this factor in the heavy chain enhancer, although there is onein the SV40 enhancer. Examples 6 and 7 describe cloning of twotranscriptional factors: NF-KB and IgNFB.

EXAMPLE 6. Cloning of putative NF-κB Experimental Procedures

λgt11-EBNA-1 Recombinant

A HinfiI-AhaII DNA fragment of the EBV genome (coordinates107,946-109,843), that contains the EBNA-1 open reading frame, wassubcloned using BamHI linkers into the BamHI site of pUC13 (pUCEBNA-1).The λgt11-EBNA-1 recombinant was constructed by inserting the 600 bpSamI-BamHI fragment of pUCEBNA-1 (EBV coordinates 109,298-109,893) intothe EcoRI site of λgt11 using an EcoRI linker (GGAATTCC). A phagerecombinant containing the EBNA-1 insert in the sense orientation wasisolated by immunoscreening with EBNA-1 antibodies (see below). In thisrecombinant, the carboxy-terminal region of EBNA-1 (191 amino acids) isfused in frame to the carboxy-terminus of β-galactosidase.

λgt11 cDNA Expression Library

The human B cells (RPMI 4265) CDNA library constructed in the expressionvector λgt11 was purchased from Clontech Laboratories, Inc. The librarycontains approximately 9×10⁵ independent clones and has an averageinsert size of 1.2 kb.

E. Coli Strains

The standard pair of λgt11 host strains, Y1090 and Y1098, were employed.The former was used to screen λgt11 recombinants and the latter togenerate λlysogens for the analysis of β-gal fusion proteins.

Plasmids

The plasmid pUCoriP1 was constructed by subcloning the EcoRI-Ncolfragment from the oriP region of the EBV genome into the SmaI site ofpUC13. This fragment contains 20 high affinity binding sites for EBNA-1.pUCoriP2 was derived from pUCoriP1 by subcloning of an orip fragment(EcoRI-BstXI) of the latter into the SmaI site of pUC13. pUCoriP2contains 11 high affinity binding sites for EBNA-1. pUCORIλ2 was made byinsertion of a synthetic binding site for the bacteriophase λO protein(AAATCCCCTAAAACGAGGGATAAA) into the SmaI site of pUC13. Thecomplementary oligonucleotides were a gift of R. MacMacken. PUCMHCI andpUCmhcI were constructed by insertion of the following oligonucleotides:##STR3## into the BamHI site of pUC13. The wild type sequence is abinding site for H2TF1 and NF-κB. pUCOCTA is a similarly constructedpUC18 derivative that contains a synthetic recognition site (ATGCAAAT)for the mammalian octamer binding protein(s). The plasmids p190H2KCAT(-190 to +5) and p138H2KCAT (-138 to +5) contain 5'-deletions of theH-2K^(b) gene promoter fused to the coding sequence for chloramphenicolacetyl transferase. All plasmid DNAs were purified by an alkaline lysisprotocol followed by two bandings in CsCl-EtBr gradients.

Binding Site Probes Competitor DNAs

The MHC, mhc1, ori and OCTA probes were generated by digesting thecorresponding pUC plasmids with EcoRI and HindIII. The resultingproducts were end-labeled with α-³² !dATP using the large fragment of E.coli DNA polymerase I. dCTP, dGTP and dTTP were included in thesereactions so as to fill in the ends of the restriction fragments. Thelabeled fragments were separated by native polyacrylamide gelelectrophoresis. The binding site fragments (60-75 bp) were eluted fromthe gel and purified by ELUTIP™ (Schleicher and Schuell) chromatography.Using high specific activity α-³² P!dATP (5000 Ci/mmol), typicallabelings yielded DNA probes with specific activities of 2-4×10⁷cpm/pmol.

To generate the orip probe, pUCoriP2 was digested with EcoRI andHindIII, and the orip fragment (˜400 bp) isolated by low melt agarosegel electrophoresis. This DNA fragment was then digested with HpaII andthe products labeled as detailed above. The smaller of the two HpaIIfragments (˜90 bp) was isolated for use as the orip probe. The MHCgprobe was prepared by digesting p190H2KCAT with XhoI was labeling asbefore. The labeled DNA was then digested with HincII and the 90 bpprobe fragment purified as before. This probe contains sequence from-190 to -100 of the upstream region of the H-2K^(b) gene.

The Δ6MHCg (-190 to +270) and Δ11MHCg (-138 to +270) competitor DNAswere prepared by digesting the plasmids, p190H2KCAT and p138H2KCAT, withXhoI and EcoRI. The H2KCAT fragments were isolated by low melt agarosegel electrophoresis.

RESULTS

Specific Detection of a λ Recombinant Expressing EBNA-1

A model system was used to test the notion that a recombinant cloneencoding a sequence-specific DNA binding protein could be specificallydetected with a recognition site probe. The Epstein-Barr virus nuclearantigen (EBNA-1) was selected as the model protein. EBNA-1 is requiredfor maintenance of the EBV genome as an autonomously replicating plasmidin human cell lines. It is also a transactivator of viral geneexpression. The carboxy-terminal region of EBNA-1 (191 amino acids) hasbeen expressed in E. coli as a fusion protein and shown to encode asequence-specific DNA binding domain. The fusion protein binds tomultiple high affinity sites at three different loci in the EBV genome.Two of these loci consitute a cis-acting element required formaintenance of the plasmid state (oriP). In the λgt11-EBNA-1 (λEB)recombinant the carboxy-terminal region of ENBA-1 was fused in frame tothe carboxy-terminus of β-galactosidase (FIG. 9). A lysogen harboringthe λEB phage conditionally expressed a β-gal-EBNA-1 fusion protein ofexpected size (approximately M.W. 145,000) that accumulated to a levelof about 1%. The DNA binding activity of the fusion protein was assayedwith a segment of orip DNA that contained two high affinity sites forEBNA-1 (FIG. 9). Extracts of λgt11 and λEB-lysogens were incubated withlabeled oriP DNA and the products resolved by native polyacrylamide gelelectrophoresis. With the λEB extract, a distinct set of protein-DNAcomplexes was observed. The formation of these complexes wasspecifically competed by an excess of plasmid DNA containing EBNA-1binding sites. Thus, the β-gal-ENBA-1 fusion protein has the expectedsequence-specific DNA binding activity.

To establish conditions for detection of EB plaques with probes of oripDNA, protein replica filters were generated from platings of the phage.These filters were screened with a variety of protocols using oriP orcontrol DNAs. Under a defined set of conditions (see ExperimentalProcedures), λEB plaques can be specifically detected using radiolabeledorip DNA. The control probe (ori) contains a high affinity binding sitefor the bacteriophage λO protein. The specific array of spots generatedby the orip probe corresponded to plaques on the master plate as well asto spots that reacted with antiserum to β-gal on the replica filter.Furthermore, in a similar experiment the orip probe did not detectcontrol λgt11 plaques. From a series of such experiments the followingconclusions were drawn; (i) the specific detection of λEB plaquesrequires a DNA probe with at least one binding site for EBNA-1 (a duplex30-mer with a consensus binding site sequence gave a signal comparableto a probe containing two or more binding sites), (ii) DNA probes longerthan 150 bp yield higher non-specific signals, (iii) the addition of anexcess of non-specific competitor DNA poly(dI-dC)-poly(dI-dC)! to thebinding solution reduces the non-specific signal, and (iv) both specificand non-specific interactions of the DNA probe with proteins on thereplica filter are reversible. In view of this latter point and the factthat non-specific interactions typically have much shorter half-livesthan the specific interactions, sequence-specific binding proteins canbe detected after a suitable wash time.

Given the ability to specifically detect λEB plaques with orip DNA,reconstruction experiments were carried out to test the sensitivity ofthe screen. In these experiments the λEB phage was mixed with an excessof control λgt11 recombinants. Relica filters generated from such mixedplatings were screened initially with orip DNA and subsequently withantibodies to EBNA-1. In an experiment where approximately 5,000 phagewere plated, with λEB being present at a frequency of 10⁻², a identicalnumber of positives (approximately 50) were detected with both orip DNAand antibody probes. In fact, the two patterns are superimposable.Furthermore the signal to noise ratio of the DNA binding site probe wasbetter than that of the antibody probe. Thus it is possible to screenfor the λEB phage with an orip DNA probe.

Screening for Mammalian Clones Encoding Sequence-Specific DNA BindingProteins

A λgt11 library of cDNAs prepared with mRNA from human B cells wasscreened using the conditions developed with λEB. The DNA probe used inthe screen contained a regulatory element from a mouse MHC class I gene(H-2K^(b), FIG. 10). This sequence (MHC) was synthesized and cloned intothe pUC polylinker. The macriptin transcriptional regulatory factorsH2TF1 and NF-κB bind with high affinity to this MHC element. In a screenof 2.5×10⁵ recombinants, two positive phage, designated λh3 and λh4,were isolated. In an autoradiogram of a filter from the primary screen,a positive spot resulted in the isolation of λh3. Partially purified λh3and λh4 phage were challenged with other DNA probes to determine iftheir detection was specific for the MHC probe. λh3 and λh4 were notdetected by the ori probe.. These phage were also not detected bylabeled pUC polylinker DNA or by a related probe (OCTA) containing arecognition site for the immunoglobulin octamer binding protein(s). Amutant MHC binding site probe (mhcl FIG. 10) was used to morestringently test the sequence-specificity of the presumptive fusionproteins. The mhcl probe did not detect either λh3 or λh4 plaques. Thesedata strongly suggested that the two phage express proteins that bindspecifically to the MHC element.

Characterization of the DNA Binding Proteins Encoded by λh3 and λh4

Direct evidence that the β-gal fusion proteins encoded by λh3 and λh4are responsible for the sequence-specific DNA binding activities wasobtained by screening Western blots with DNA and antibody probes.Lysogens of λgt11, λh3 and λh4 were isolated and induced to generatehigh levels of their respective β-gal proteins. Western blots ofproteins from induced lysogens were prepared and the immobilizedproteins were briefly denatured with 6M guanidine and then allowed torenature (see Experimental Procedures above). This treatment increasedthe recovery of active molecules. Two equivalent transfers wereinitially probed with either the MHC element or the OCTA control DNA. Aset of four bands specific to the MHC probe and the λh3, λh4 tracks wasobserved. The two largest species of this set are labeled P1 and P2. Thesame transfers were then probed with antibodies to β-gal. A pair ofnovel fusion protein bands was observed with each of the two recombinantlysogens. These bands corresponded to the species P1 and P2 detectedwith the MHC probe. This shows that λh3 and λh4 encode β-gal fusionproteins which bind specifically to the MHC element DNA. The two phagemay be identical since they encode the same size fusion proteins. P1(approximate m.w. 160,000) probably represents the full length fusionprotein whereas P2 is a presumptive proteolytic cleavage product. Sincethe β-gal portion of this fusion polypeptide has a molecular weight ofapproximately 120,000, the cDNA encoded portion must have a molecularweight of 40,000.

A gel electrophoresis DNA binding assay was used to confirm the sequencespecificity of the λh3 and λh4 fusion proteins as well as to betterdefine their recognition properties. Extracts derived from the λgt11,λh3 and λh4 lysogens were assayed, with the MHC probe. A novel DNAbinding activity was detected specifically in extracts of the λh3 andλh4 lysogens. This activity was IPTG inducible indicating that it was aproduct of the lacZ fusion gene. A competition assay indicated that theactivity represented a sequence-specific DNA binding protein. Two 5'deletion mutants of the H-2K^(b) genomic sequence was used as competitorDNAs. The segment 6MHCg extends to 190 nucleotides upstream of thetranscription start site and contains the MHC sequence element. Thesegment Δ11MHCg, on the other hand, only contains 138 nucleotides ofsequences upstream of the initiation site and therefore lacks the MHCelement. Increasing amounts of Δ6MHCg specifically competed for thebinding of the λh3 fusion protein to the MHC element oligonucleotideprobe while the control Δ11MHCg did not compete. It should be noted thatthe sequences flanking the MHC element in the probe used for the initialscreening, the cloned oligonucleotide, are totally difference from thesequences flanking the same element in the genomic probe, Δ6MHCg.Therefore, the fusion protein appears to exclusively recognize thecommon MHC element. This was confirmed by a direct DNA binding assaywith a genomic sequence probe (MHCg) containing the MHC element. Boththe oligonucleotide (MHC) and genomic (MHCg) probes gave rise tosimilarly migrating complexes. Furthermore, a double base substitutionmutant (mhcl, FIG. 10) abolished recognition by the fusion protein. Themutant sequence contains a transverion in each half of the symmetric MHCelement. These changes destroy the symmetry of the element and abolishbinding by either H2TF1 or NF-κB.

The immunoglobulin κ chain gene enhancer contains a binding site (EN)for NF-κB. This site is related in sequence to the MHC element but isrecognized by H2TF1 with a 10 to 20 fold lower affinity (FIG. 10). Amutant κ enhancer (κEN) has been characterized both in vivo and invitro. This mutant sequence has no B cell specific enhancer activity andis not bound by NF-κB. The mutant contains clustered base substitutionsand an insertion of a base pair in one of the two symmetric half sites(FIG. 10). The binding of the λh3 fusion protein to the wild typeκ-element and the mutant version was tested. The κEN probe generated acomplex with a mobility similar to those obtained with the MHC probes.No specific complex was formed with the mutant κ-enhancer DNA.Experiments in which the MHC and κ-enhancer binding sites were testedfor competition with binding of the MHC probe showed that the fusionprotein bind with 2-5 fold higher affinity to the MHC site. The κEN sitediffers, in part, from the MHC site by the substitution of two adenineresidues for guanine residues. As discussed below, these guanineresidues are probably contacted by the fusion.

The contacts of the fusion protein with the MHC element were probedchemically by modification of the DNA with dimethylsulfate. Afterpartial methylation at purine residues, the modified probe was used inthe gel electrophoresis DNA binding assay. Free (F) and bound (B) probeDNA was recovered, subjected to chemical cleavage at methylatedinterference experiment. On both the coding and non-coding strandsstrong interference was detected when any of central guanine residues ofeach putative half site was modified at the N-7 position in the majorgroove. Weaker interference was observed when the external guanineresidue in either putative half site was similarly modified. Thus thefusion protein appears to symmetrically contact the MHC element in amanner similar to both H2TF1 and NF-κB.

Hybridization Analysis with the cDNA Segment of the Recombinant Phage

The recombinant phage λh3 and λh4 contain cross-hybridizing andequivalent size (approximately 1 b) cDNA segments. The inserts also haveindistinguishable restriction maps and therefore appear to be identical.Southern blot hybridization confirmed that these cDNA segments arehomologous to sequences in the human genome. The patterns ofhybridization to restriction digests of genomic DNAs of various humancell lines are identical. Furthermore, the fact that restriction digestswith Bam HI (no site in cDNA) and Pst I (on site in cDNA) both generatetwo prominent bands suggests that the cDNAs are derived from a singlecopy gene. A similarly simple hybridization pattern is observed onprobing the mouse and rat genomes.

The expression of the human gene was analyzed by Northern blothybridization. A single, large transcript (approximately 10 kb) wasobserved with polyA(+) RNA from both B (X50-7) and non-B human cells(HeLa). This transcript is moderately abundant in both cell types. Sincethe cDNA library was constructed by oligo dT priming, we were probablyfortunate to obtain the coding region for the DNA binding domain withinthe 1 kb segments of the recombinant phage. However, this onlyillustrates the power of the screening strategy for the isolation ofclones encoding sequence-specific DNA binding domains.

Discussion

A novel strategy is disclosed for the molecular cloning of genesencoding sequence-specific DNA binding proteins. This strategy can beused to isolate genes specifying mammalian transcription regulatoryproteins. An important step in this approach is the detection ofbacterial clones synthesizing significant levels of a sequence-specificDNA binding protein by screening with a labeled DNA binding site probe.This approach is similar to that previously developed for the isolationof genes by screening recombinant libraries with antibodies specific fora given protein. In fact, the phage expression vector, λgt11, developedpreviously for immunological screening can be in this approach.

The feasibility of the strategy was established by the specificdetection of a phage recombinant, λEB, encoding a β-gal-EBNA-1 fusionpolypeptide with orip DNA. Conditions have also been developed for theselective detection of E. coli colonies expressing high levels of EBNA-1or the bacteriophage λO protein with their respective binding site DNAs.In these cases, a plasmid expression vector was employed. Using theconditions developed with λEB, we have screened phage cDNA librarieswith three difference DNA probes. Screening with a probe containing theH2TF1 site in the MHC class I gene H-2K^(b) led to the isolation of twoidentical clones that specify a putative transcription regulatoryprotein (properties discussed below). In similar screens with two otherDNA probes, positive recombinant phage were also isolated at a frequencyof approximately 1/100,000. However, the DNA binding proteins encoded bythese phage do not appear to recognize specific sequence elements butrather to bind sequence nonspecifically to either single strand ordouble strand DNA. Although detection of these types of clonesrepresented a troublesome background in this study their isolationsuggests that recombinants encoding different types of DNA bindingproteins can be detected by such functional screens of expressionlibraries. In future screens for recombinants encoding site-specific DNAbinding proteins, the detection of these other types of clones might beselectively suppressed by inclusion of a non-specific competitor DNAthat is structurally more similar to the probe thanpoly(dI-dC)-poly(dI-dC).

The prospects for the isolation of other cDNAs encodingsequence-specific binding protein by this strategy can be assessed byexamining the three assumptions on which it is based: (i) functionalexpression of the DNA binding domain of the desired protein in E. coli,(ii) a strong and selective interaction of the binding domain and itsrecognition site, and (iii) high level expression of the DNA bindingdomain. A number of eukaryotic sequence-specific DNA binding proteinshave been functionally expressed in E. coli. These include the proteinsGAL4, GCN4 and MAT 2 of yeast, ftz of Drosophila, TFIIIA of Xenopus, E2of the bovine papilloma virus and EBNA-1 of the Epstein Barr Virus. Inmost cases, the functional DNA binding domain is contained within ashort tract of amino acids. Thus, it is reasonable to expect thefunctional expression in E. coli of the sequence- specific DNA bindingdomain of most eukaryotic regulatory proteins. The equilibriumassociation constants of site-specific DNA binding proteins range overmany orders of magnitude (10⁷ -0¹² M). The following analysis suggeststhat successful screening may be restricted to proteins with relativelyhigh binding constants. If a regulatory protein has an associationconstant of 10¹⁰ M, then under the screening conditions (the DNA probeis in excess and at a concentration of (˜10¹⁰ M) approximately half ofthe active molecules on the filter will have DNA bound. Since thefilters are subsequently washed for 30 minutes, the fraction ofprotein-DNA complexes that remain will be determined by theirdissociation rate constant. Assuming a diffusion limited associationrate constant of 10⁷ M⁻¹ S⁻¹, the dissociation rate constant will be10⁻³ S⁻¹. Such a protein-DNA complex will have a half life ofapproximately 15 minutes. Thus only a quarter of the protein-DNAcomplexes will survive the 30 minute wash. For a binding constant of 10⁹M, only about a tenth of the active protein molecules will have DNAbound and much of this signal will be lost, since the half-life of thesecomplexes is approximately 1.5 minutes. Isolation of recombinantsencoding proteins with binding constants of 10⁹ or lower may be possiblegiven that the binding of probe to less than 1% of the total fusionprotein within a plaque can be detected. The sensitivity of the currentmethodology for low affinity proteins could be significantly enhanced bycovalent stabilization of protein-DNA complexes. This might beaccomplished by procedures such as UV-irradiation of pre-formedcomplexes. Since the binding constants of regulatory proteins aredependent on ionic strength, temperature and pH, these factors mightalso be manipulated to enhance detection.

The successful detection of λEB and λh3 recombinants with DNA bindingsite probes required high level expression of their fusion proteins. Inboth cases, the fusion proteins accumulate, after induction, to a levelof about 1% of total cellular protein. This level of recombinant proteinexpression is typical of λgt11 as well as other E. coli vectors. Thestrategy of cloning a gene on the basis of specific detection of itsfunctional recombinant product in E. coli has considered potential.Indeed, while our work was in progress, this approach was used by otherto isolate clones encoding a peptide acetyltransferase and acalmodulin-binding protein. Direct screening of clones encodingrecombinant protein products has also been used to isolate rasGTP-binding mutants.

The λh3 recombinant expresses a β-gal fusion protein that recognizesrelated transcription control elements in the enhancers of the MHC classI and immunoglobulin κ-chain genes (see FIG. 10 for sequences). Thisprotein also binds a similar element in the SV40 enhancer 72bp repeat.Furthermore, there are two putative binding sites in the long terminalrepeat (LRT) of the HIV genome (FIG. 10). One of these is identical tothe site in the SV40 enhancer and therefore should be recognized by thefusion protein. The existence of a clone such as λh3 was anticipatedsince it had previously been shown that a common factor, NF-κB, binds tothe three related elements in the enhancer, the SV40 72 bp repeat an theHIV-LTR. Interestingly, these three binding sites are more closelyrelated to one another than they are to the MHC site (FIG. 10). Theformer set can be viewed as variants of the MHC site which exhibitsperfect two-fold symmetry. It should be noted that the pUC polylinkercontains the sequence, CGGGGA, which is a variant of one of thesymmetric halves (TGGGGA) of the MHC element. The fusion protein doesnot bind with detectably affinity to the pUC polylinker. Thus, a highaffinity interacter appears to require both symmetric halves.

Even though the above control elements represent quite similarsequences, they function in very different regulatory capacities. TheMHC element is a component of an enhancer that functions in a variety ofcell types that express MHC class I genes. The κ-element, on the otherhand, is a component of a cell-type specific enhancer that functionsonly in B cells. The activity of this enhancer is induced in pre-B cellsupon their differentiation into mature B lymphocytes. Suchdifferentiation, in vitro, is accompanied by transcriptional activationof the chain gene. The κ-element appears to dictate the B cellspecificity of the κ-enhancer. The different modes of functioning of theMHC and κ-elements are correlated with the properties of theircorresponding recognition factors, H2TF1 and NF-κB. H2TF1 activity isdetected in a variety of differentiated cell types and this proteinappears to stimulate MHC class I gene transcription approximately10-fold. On the other hand, NF-κB activity is detected only a mature Bcells. In addition, this activity is induced during differentiation ofpre-B cells to mature lymphocytes. Finally, NF-κB activity is alsoinduced by phorbol ester treatment of non-B cell lines (HeLa, Jurkat).In the case of Jurkat cells, a T4⁺ human T cell line, NF-κB appears tostimulate the transcriptional activity of the HIV-LTR. It should benoted that induction of NF-κB in non-B cells does not require newprotein synthesis. Thus the protein for NF-κB must exist in cells beforeinduction and the activated by a post-translational modification.

The DNA binding properties of the fusion protein encoded by therecombinant λh3 overlap those of H2TFl or NF-κB. Mutants of the MHC andκ-elements that are not recognized by H2TFl or NF-κB are also not boundby the fusion protein. The recombinant protein binds the MHC element DNAwith 2-5 fold higher affinity than the κ-element. In this regard, thefusion protein has relative affinities intermediate between those ofH2TF1 and NF-κB. H2TF1 binds the MHC element with 10- to 20-fold higheraffinity than the κ-element while NF-κB recognizes both elements withroughly equivalent affinity. This intermediate relationship is alsoobserved in the comparison of the methylation interference patterns ofthe three DNA binding activities. Methylation of any of the central sixguanine residues in the MHC site strongly interfers with the binding ofall three activities. Methylation at either of the two external guaninespartially interferes with recognition by the fusion protein. Incontrast, H2TF1 binding is strongly suppressed upon methylation ofeither of these residues while NF-κB binding shows little perturbation,upon this modification. This analysis of the three DNA bindingactivities is limited by the use of cell extracts and not purifiedproteins. Furthermore, the properties of a recombinant protein may bedifferent from those of its native counterpart. Thus, it is not possibleto be definitively relate the protein encoded by λh3 to either H2TF1 orNF-κB.

Antibodies raised against the λh3 fusion protein will be useful inclarifying its structural relationship with H2TF1 and NF-κB. Adefinitive relationship will emerge from a comparison of the deducedamino acid sequence of the cDNA and the protein sequences of H2TF1 andNF-κB. It should be noted that in terms of protein expression, bothH2TF1 and NF-κB are present in a wide variety of mammalian cells.Furthermore, the DNA binding specificities of these two factors areremarkably similar. These facts as well as the observations that thecDNA in λh3 hybridizes to a single copy gene and to a single mRNA inboth B and non-B cells suggest that all three binding activities may beproducts of the same gene. This hypothesis would imply that H2TF1 andNF-κB represent alternative modifications of a common protein.

EXAMPLE 7 Cloning of the IgNFB Gene Methods

DNA Sequencing

DNA sequencing was performed on double stranded plasmid DNA templatesaccording to the Sanger dideoxy-nucleotide protocol as modified byUnited States Biochemical for use with bacteriophage T7 DNA polymerase(Sequenase). The entire sequence was confirmed by sequencing theopposite strand and in the GC-rich regions by sequencing according toMaxam and Gilbert (Methods Enzymol., 65: 449-560, (1980)

Plasmid Constructions

cDNA's were subcloned from λgt11 to pGEM4 (Promega), and these plasmidswere used for DNA sequence analysis and in vitro transcription. PlasmidpBS-ATG was kindly provided by H. Singh and K. LeClair and wasconstructed by ligating a 27 bp long oligonucleotide containing an ATGcodon surrounded by the appropriate boxes for efficient initiation,TGCACACCATGGCCATCGATATCGATC, into the Pstl site of pBS±Bluescroptplasmid (Stratagene). The expression vector pBS-ATG-oct-2 depicted inFIG. 11A was designed for transcription and translation in vitro and wasconstructed by cleaving pBS-ATG with Smal and ligating the blunt-endedEcoRI 1.2 kb cDNA fragment from plasmid 3-1 (position 655 to 1710 inFIG. 11A).

In Vitro Transcription/Translation

In vitro transcription and translation reactoins were performed asrecommended by the manufacturer (Promega).

DNA Binding Assay

The EcoRI/HindIII 50 bp fragment containing the wild type octanucleotidesequence ATGCAAAT in the BamHI site of pUC18 polylinker was ³² P-labeled(50,000 cpm/ng) and 1 ng DNA probe was incubated with 1 μl of thereacted/unreacted reticulocyte lysate. The binding reactions wereincubated at room temperature for 30 minutes and contained 10 mM TrisHCl pH7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA pH8, 5% glycerol, 25 μl/mlsonicated denatured calf thymus DNA in 2.5 μl/ml sonicated native calfthymus DNA as nonspecific competitors. The complexes were resolved byelectrophoresis in 4% polyacrylamide gel (acrylamide:bisacrylamideweight ratio of 20:1), containing as buffer 25 mM Tris HCl pH8.5, 190 mMglycine, 1 mM EDTA buffer as previouly described (Singh et al., Nature319: 154-158 (1986)).

Purification of NF-A2

NF-A2 was purified to >90% homogeneity from nuclear extracts derivedfrom the human Burkett's lymphoma cell line, BJAB. Purification wasaccomplished by sequential fractionation on Zetachrom QAE discs (CunoInc.), heparin sepharose (Pharmacia), ssDNA cellulose (Pharmacia), andon a DNA affinity column which contained an immobilized double straded(ds) segment containing the octanucleotide seuqnce. In vitro translated,³⁵ S-methionine-labeled, oct-2 protein was purified by chromatography ondsDNA cellulose followed by affinity chromatography on theoctanucleotide DNA affinity column.

Tryptic Digestions of NF-A1 and oct-2 Protein

Tryptic digests were performed at room temperature in a bufferconsisting of 20 mM Hepes,KOH, pH 7.9, 20% glycerol, 0.5M KC1, 0.2 mMEDTA, 0.5 MM DTT. Aliquots of purified NF-A2 (˜250 ng) or of affinitypurified oct-2 protein (90,000 cpm) were incubated with varying amountsof trypsin (affinity incubated with varying amounts of trypsin (affinitypurified trypsin was a gift of Dan Doering). After 60 minutes, reactionswere terminated by the addition of 2.5 volumes of SDS-PAGE sample bufferand were boiled for 5 minutes. The tryptic digestes were resolved on 10%polyacrylamide gels. Tryptic fragments of NF-A2 were visualized bysilver-staining. Tryptic fragments of ³⁵ S-methionine labeled oct-2protein were visualized by autoradiography after treatment of the gelwith En³ hance (Dupont).

To clone the gene encoding the lymphoid-specific octamer bindingprotein, IgNF-B (NF-A2), a randomly primed, non-size selected cDNAlibrary in λgt11 was constructed using cytoplasmic poly (A)-containingmRNA from a human B cell lymphoma cell line, BJAB. We had previouslyobserved that this cell line contained a particularly large amount ofNF-A2 when 28 lymphoid cell lines were surveyed. By randomly priming thecDNA synthesis we expected to obtain recombinant phage encoding theoctamer motif binding domain even if that domain was encoded by the 5'end of a long mRNA. The randomly primed cDNA library in λgt11 wasgenerated by standard methods (Gubler, U. and Hoffman, B. J., Genes25:263-269 (1983)). Random hexamers (Pharmacia) were used to prime thefirst strand cDNA synthesis. The unamplified library contained 500,000recombinants. This library was screened by the method described aboveusing a radiolabelled DNA probe consisting of four copies, in directorientation, of a 26 bp oligonucleotide derived from the Vk41 promoter.The probe was constructed by cloning four copies of the oligonucleotidein direct orientation into the BamHl site of the pUC polylinker andradiolabelling the 112 bp Sma1-Xba1 fragment. The library was screenedwith the tetramer probe (at 1×10⁶ cpm/ml) as described above for thecloning of NF-κB with the following modification. Previous screens usingpoly(dI-dC)-poly(dI-dC) as the nonspecific competitor DNA yieldedrecombinant phage encoding single stranded DNA binding proteins. Thesignal from these phage but not phage encoding sequence-specific DNAbinding proteins could be efficiently competed with denatured calfthymas DNA (5 μg/ml) and therefore this nonspecific competitor wassubstituted for poly(dI-dC)-poly(dI-dC) in all subsequent screens.

From a primary screen of 450,000 phage plaques, three plaques wereisolated which bound this tetramer probe. Two of these phage, phage 3and phage 5, were found to give plaques that bound specifically to thetetramer probe in that they did not bind DNA probes which lacked theoctamer motif. These two phage bound probes containing one copy of the κpromoter octamer motif with a much lower affinity than they bound thetetramer probe. Even when four-fold more monomer probe was used thentetramer probe, the tetramer probe still gave a greater signalsuggesting that the better binding of the tetramer probe was not merelya result of increasing the molar concentration of binding sites in thescreen. Certainly in the case of phage 5, which showed dramaticallybetter binding to the tetramer probe, it seems most likely that thetetramer probe was able to bind simultaneously to multiple phage fusionproteins on the filter. This multipoint attachment would be expected todramatically decrease the dissociation rate and thus, increase theavidity of the interaction. Genes encoding DNA binding proteins withrelatively low binding affinities could be cloned by screening λgt11expression libraries with such multimer probes.

The specificity of the DNA binding proteins encoded by the recombinantphage was investigated by preparing extracts of induced phage lysogens.Lysogen extracts from both phages bound to the tetramer probe in amobility shift assay whereas lysogen extracts from non-recombinant λgt11showed no binding to this probe. Only the phage 3 extract bound stronglyto the κ promoter probe. Because the inserts of phage 3 and phage 5 (1.2kb and 0.45 kb in size, respectively) were found to cross-hybridize bySouthern blotting analysis, phage 3 was chosen for further analysis.

Phage 3 encoded an octamer binding protein as demonstrated by acompetition mobility shift assay in which the lysogen extract was boundto the κ promoter probe in the presence of competing unlabelled DNAfragments containing either the wild type or mutant octamer motifs.Phage lysogen extracts were prepared as described above for NK-κBcloning. The extracts were assayed in a mobility shift assay asdescribed above using the octamer-containing PvuII-EcoR1 fragment frompSPIgVk as the radiolabelled probe. Binding reactions were carried outin the absence or presence of 24ηg of cold competitor DNA containing nooctamer motif, the wild type octamer motif or mutant octamer motifs asdescribed.

The wild type octamer motif competed efficiently for binding but theoctamer motifs containing point mutations either did not compete orcompeted less well than the wild type motif. In fact, the two mutantswhich showed slight competition for the binding of the lysogen protein,TCATTTCCAT and ATATTGCAT, were the only mutants which somewhat competedthe binding of NF-A1 and NF-A2 in a WEHI 231 nuclear extract.

The phage-encoded octamer binding protein was further compared to NF-A1and NF-A2 using a methylation interference footprinting assay.Methylation interference was performed as described using the non-codingstrand of the octamer-containing PvuII-EcoR1 fragment of pSPlgVk asradiolabelled probes. The probes were partially methylated and used inpreparative mobility shift DNA binding assays. DNA present in the boundbands (NF-A1 and NF-A2 bands from a nuclear extract from the BJAB cellline or phage 3 lysogen extract bound band) or free bands was isolated,cleaved at the modified purine residues and subjected to denaturingpolyacrylamide gel electrophoresis. The footprint obtained using thelysogen extract was centered over the octamer motif and was very similarto the footprints of NF-A1 and NF-A2 from a BJAB nuclear extract andfrom a WEHI 231 nuclear extract (see above). Minor differences were seenbetween the footprints of the lysogen and nuclear extract proteins whichcould reflect changes in affinity and/or specificty of DNA binding as aresult of fusion of the insert-encoded octamer binding protein withβ-galactosidase. Alternatively, the phage insert could encode an octamerbinding protein distinct from NF-A1 and NF-A2.

The phage-encoded β-galactosidase fusion protein was directly shown tobe the octamer binding protein in the phage lysogen extracts. Phagelysogen extracts were subjected to SDS polyacrylamide gelelectrophoresis and transferred to nitrocellulose filters. After adenaturation/renaturation procedure (Celenza, J. L. and Carlson, M.Science 233:1175-1180 (1986)), the filters were probed with either theradiolabelled octamer-containing tetramer probe (OCTA) or a non-specificDNA probe (pUC). The OCTA probe specifically bound to theβ-galactosidase fusion proteins of phage 3 and phage 5 to a much greaterextent than the pUC probe, thus formally showing that the octamerbinding activity was encoded by the phage inserts. The apparentmolecular weights of the largest fusion proteins of phage 3 and phage 5lysogens are consistent with the entire phage inserts contributingcoding sequences to the fusion proteins. Prototeolysis was presumed toaccount for the heterogeneity in apparent molecular weight of the fusionproteins.

The insert of phage 3, which defines what we term the OCT-2 gene, wasused in a Southern blot analysis to probe human and mouse genomic DNAdigested with several restriction enzymes. Restriction enzyme digestedgenomic DNA was electrophoresed through a 1% agarose gel and transferredto Zetabind (CUNO Laboratory, Inc.) by standard techniques (Maniatis,T., Frisch, E. F. and Sambrook, J. Molecular Cloning. A LaboratoryManual. Cold Spring Harbor Laboratory Press, New York (1982)). The phage3 insert was radiolabelled by randomly primed synthesis usinghexanucleotides (Pharmacia). Following standard prehybridizationhigh-stringency hybridization (Maniatis, supra.) with the OCT-2 probethe filters were washed with 0.2× SSC, 0.1% SDS or 2× SSC, 0.1% SDS.

One or two bands were observed in each restriction enzyme digest whichis consistent with OCT-2 being a single genetic locus. No rearrangementsor amplifications of the gene were observed in a survey of 8 lymphoidand non-lymphoid cells lines including BJAB. The strength of the signalon the mouse Southern blot at high stringency suggested that the gene ishighly conserved between human and mouse.

The oct-2 cDNA segment (1.2 kb) of phage 3 was used to identifyadditional overlapping recombiants in the same library. One of thesephage (pass-3) contained a 1.8 kb DNA insert. Sequence analysis of thecDNA segment in the original λgt11 phage (3-1) revealed a long openreading frame (ORF) which was ended with multiple nonsense codons at its3' terminus. Sequence analysis of the pass-3 segment yielded anidentical sequence through the open reading frame but an abrupttransition to a novel sequence occurred at the C-terminue (FIG. 18B; seebelow). The N terminus of the open reading frame in both of these cDNAsegments was not represented in the cDNA inserts. Additional recombinatsfrom the λgt11 library were identified by scrrening with a probe fromthe NOterminal portionof the pass-3 segment. This resulted in theisolationof a 0.75 kb cDNA segment (pass-5.5) whose sequence extendedthe N-terminal portion o the previously identified open readign frame.In this cDNA segment, a nonsense codon is foudn 36 pb upstream of thefirst AUG in the open frame. The sequence context of this AUG confomswell to that expected for an initiation codon (Kozak, Cell 44: 283-292(1986)). Two other AUG codons occur at positions 6 and 13 in the readingframe. Each of these also has an excellent context for initiation. The Nterminus of the protein has been arbitrarily assigned to the 5'-most AUGcodon. The cDNA sequence extends 66 bases 5' from this positon but thetotal length of the 5' untranslated region has not been determined.

The sequences of pass-5.5, pass-3 and 3-1 were combined to form an openreading frame encoding a protein of 466 amino acids in length as shownin FIGS. 11A-11C. FIGS. 11A-11C the amino-acid sequence of oct-2 proteindepicted in plain capital letters. cDNA-clone pass-5.5 spans fromposition 1 (5' end) to position 750 (3' end). cDNA clone pass-3 5' endand 3' end are respectively at position 92 in FIG. 11A and 1847 in FIG.11B. cDNA clone 3-1 starts at position 650 and ends at position 1710.The nucleotide sequence shown in panel A was reconstructed by mergingthe DNA sequences from clone pass-5.5 from position 1 to 100, from clonepass-3 from 100 to 660 and from clone 3-1 from position 660-1710.Extensive nucleotide sequence overlaps were available to allowunequivocal merges. Sequence of protein encoded by the longoverlappingopen reading frame (LORF, 277aa) is shown in italic letters.Wavy arrows delimit the glutamine (Q)-rich, glutamic and aspartic(E/D)-rich and glycine (G)-rich regions, respectively. Solid arrowsdelimt the helix-turn-helix motif. Boxed leucine (L) residues are spacedexactly by seven residues. Vertical arrow indicates the position wherethe nucleotide seuqnece diverges with that shown in panel B. Starsindicate stop codons.

FIG. 11B shows the nucleotide sequence of the 3' terminus and redictedamino acid sequence of the C-terminus derived from clone pass-3. Thecode is the same as in A and the vertical arrow denotes the divergencepoint.

FIG. 11C is a schematic representation of the amino acid sequencededuced from oct-2 gene derived cDNA. The code is as in panel A. The DNAbinding domain is depicted as DNA and the region containing the fourregularly spaced L residues is boxed-in. LORF stands for long openreading frame, N stands for N-terminus and C for COOH-terminus.

Data presented below suggests that this ORF encodes one form of NF-A2(oct-2). The amino acid sequence of oct-2 has several interestingfeatures (FIG. 11C). It contains three glutamine (Q) rich blocks(ranging from 50% of Q content) in teh N-terminal part of thepolypeptide, beginning at nucleotide postions 376, 448 and 502, and acomparably acidic region aspartic (E) or glutamic (D) amino acids!between postions 648 and 678. Clusters of Q resideus as well as E or Damino acids have been described previously in many transcriptionfactors. Such acidic regions in other factors have been shown to beimportant in activation of transcroption (Gill and Ptashne, Cell 51:121-126 1987; Hope et al., Nature 333: 635-640 (1988)).

The region of oct-2 responsible for sequence-specific DNA bidnign,depicted "DNA", is discussed below. Downstream of this position is aseries of four leucine residues separated by exactly seven amino acids(position 1227 to 1293 in FIG. 11A). A similar configuration of leucineresidues in the transcription factor C/EBP has been suggested to form anamphipathic α-helical structure where the leucine residues are arrangedalong one side of the helix. Two such helices are throught to interactby a "leucine zipper" mechanism generating a dimeric protein(Handschultz et al., Science 240: 1759-1764 (1988); Landschultz et al.,Genes & Development 2: 786-800 (1988)).

Consistent with this sugestion, no helix disrupting proline residue ispresent in oct-2 in the 22 amino acid tract defined by the fourleucines. However, unlike the first example of a leucine zipper",protein C/EBP, the potential α-helical region in oct-2 does not possessa high density of paried charged residues which could stabilize thestructure. Also, unlike the C/EBP protein, which binds DNA specificallyas a homodimer probably by pairing through the "leucine zipper", theoct-2 protein appears to specifically bind DNA as a monomer. It isinteresting to speculate that the "leucine zipper" region of oct-2 mightbe important for interaction with other proteins as there is no obviousreason to restrict the binding of such a structure to self-recognition.

Searches for sequence similariites in the GenBank library revealed thata region of the oct-2 protein from position 952 to 1135 was distantlyrelated to a family of proteins containing homeoboxes. The 60-residuehomeobox domain is highly conserved among 16 examples in differentDrosophila genes (Gehering, Science 236: 1245-1252 (1987)). This levelof conservation extends to homeobox sequences found in vertebrates andworms. Among this total family, nine of the 60 residues are invariant.The oct-2 protein only contains six of these nine residues and four ofthese six sites are clustered in the subregion of the homeobox thoughtto be related to the helix-turn-helix structure see FIG. 13. As shown inFIG. 13, a 60 amino acid region of oct-2 contains 30% identity with theprototype homeobox sequence in the Antennapedia (Antp) protein.

FIG. 13 shows the amino acid sequence alignment of the DNA bindingdomain of oct-2 factor with homeoboxes from Antp. (Schneuwly et al.,EMBO J. 5: 733-739 (1986), cut (Blochlinger, Nature 333:629-635 (1988),en (Poole et al., Cell 40: 37-43 (1985), proteins (boxed-in amino acidsequences from the S. cerevisae proteins Matal (Miller, EMBO J. #:1061-1065 (1984), Matα2 (Astell et al., Cell 53: 339-340 (1988) and C.elegans protein mec-3 (Way and Chalfie, Cell 54: 5-16 (1988). The nineinvariant residues in canonical homeobox sequences Atnp, cut, and en arelisted below the boxed-in amino acid sequences and shown in bold printif present in the amino acid sequences. The stars indicate thehydrophobic amino acids that are critical for the protein to maintainthe helix-turn-helix structure (Pabo and Sauer, 1984). Solid arrowsdelimit the helix-turn-helix domain.

That the homeobox specifies a sequence-specific DNA binding domain ismost strongly argued by its homology with the DNA binding domain of theyeast mating regulatory protein, MATα (Astell et al., Cell 27: 15-23(1981); Scott and Weiner, Proc. Natl. Acad. Sci. USA 81: 4115-4119(1984)), which also has homology through this subregion of the homeoboxbut does not conserve the other invariant of the homeobox. Thehomologous regions in these proteins can be folded into ahelix-turn-helix structure similar to that first identified in thestructural analysis of phage λ repressor (for a review see Pabo andSauer, Ann. Rev. Biochem. 53: 293-321 (1984)). A prediction of the mostprobable secondary structure of oct-2 also revealed a helix-turn-helixstructure between the residues of isoleucine (position 1041) andcysteine (position 1090). Thus, by analogy, we propose that this regionof oct-2 specifies the sequence-specified binding of the protein.

As mentioned above, sequences at the 3' end of the pass-3 recombinantabruptly diverged from that of recombinant 3-1 at the position (1463) ofits termination codon (see vertical arrow in FIG. 11B). The substitutedsequences in the second recombinant, pass-3, extended the reading frameof the oct-2 realted protein by an additional 16 amino acids. To ruleout a possible artifactual sequence generated by the insertion offragment during construction of the cDNA library, total polyA(+) RNAfrom the BJAB cell line was analyzed by Northern blot with a DNAfragment from the novel 3' terminal portionof the pass-3 cDNA. Thisspecific probe hydribized only to the two fastest migrating mRNAs of thetotal family of six mRNAs which were detected by hybridization with thetotal 3-1 cDNA. A similar specific probe was excised from the 3'terminus of the 3-1 cDNA. In contrast, this probe only hybridized to thetwo slowest migrating mRNAs in the total family of six. This suggeststhat the two CDNA segments correspond to different populations of oct-2mRNAs.

The proteins encoded by the two cDNAs should only differ at their Cterminus by 16 amino acids or approximately 1.5 kD. In vitrotranscription/translation of subfragments of the 3-1 and pass-3recombinants was used to confirm this prediction. Fragments representingthe 3' portions of 3-1 and pass-3 were subcloned into the expressionplasmid pBS-ATG. The resulting plasmid DNAs were transcribed withbacteriophage T7 RNApolymerase and were subsequently translated in areticulocyte system. The resulting polypeptides migrated with themobilities of the anticpated molecular weights 34kD and 32.4 kD. thepolypeptide from the pass-3 cDNA was 1.6 kD larger than that fromt he3-1 CDNA. Both polypeptides specifically bound a probe containing theoctanucleotide sequence, producing a readily detectable DNA-proteincomplex in the gel mobility assay. This suggests that the oct-2 gene isexpressed as a family of polypeptides in B-cells.

The potential significance of these additional 16 amino acids isunclear. These two cDNAs almost certainly differ by alternative splicingpatterns of RNA transcribed from the oct-2 gene. Furthermore, it islikely that the oct-2 gene encodes a more diverse set of mRNAs thanthose partially defined by these two cDNAs. Six different length mRNAsare produced at significant levels in mature B cells. The relativeamounts of these mRNAs vary between pre-B, B and plasma cell lines(Staudt et al., Science 241: 577-580 (1988)). This population couldreflect variations in sites of initiation of transcription and ofpolyadenylation as well as further differences in splicing patterns.

The expression of the OCT-2 gene was assessed by Northern blot analysisof mRNA from 13 lymphoid and non-lymphoid cell lines and was found to bepredominantly restricted to lymphoid cells. Poly(A)--containing mRNA (3μg, or 20 μg) or total mRNA (30 μg) was analyzed from the following celllines. 1. NIH 3T3: mouse fibroblast; 2. 38B9: mouse pre-B cell line; 3.WEHI 231: mouse mature B cell line; 4. A431: human epidermal cell line;5. U1242: human glioma cell line; 6. RB27: human retinoblastoma cellline; 7. Jurkat: human T cell line; 8. Namalwa: human mature B cellline; 9. BJAB: human mature B cell line (poly(A)-containing mRNA); 10.BJAB (total mRNA); 11. Hut78: human T cell line; 12. HeLa: humancervical carcinoma cell line; 13. EL4: mouse T cell line. mRNA waselectrophoresed through a formaldehyde-containing 1.3% agarose gel andtransfered to a nitrocellulose filter by standard techniques (Maniatis,supra.). Following prehybridization, the filter was hybridized at highstringency with radio-labelled OCT-2 probe (above). The filter waswashed in 0.2×SSC, 0.1% SDS at 68° C. and autoradiographed with anintensifying screen at -70° C. for 24 hrs. The filter was stripped bywashing in 50% formamide, 10 mM Tris (pH 7.4), 1 mM EDTA at 68° C. for 1hr. and rehybridized with a radiolabelled rat alpha tubulin cDNA probe(Lemischka, I. R., Farmer, S., Racaniello, V. R. and Sharp, P. A., J.Mol. Biol. 151:101-120 (1981)) to control for the amount of mRNA loaded.

All five B lymphoma cell lines, including pre-B and mature B cell lines,and one of three T lymphoma cell lines expressed a family of 6transcripts. Of the five non-lymphoid cell lines tested, only a gliomacell line, U1242, showed detectable expression of this gene. Even at lowstringency we were unable to detect a transcript present in all celllines which might correspond to NF-A1. The various transcripts,estimated to be 7.2 kb, 5.8 kb, 5.4 kb, 3.7 kb, 3.1 kb and 1.2 kb long,were expressed in somewhat varying amounts relative to each other in thepositive cell lines. Whether these transcripts represent alternativemRNA splicing or highly specific mRNA degradation remains to bedetermined. In this regard, it is interesting that highly purifiedpreparations of NF-A2 consist of three or more major polypeptides withdistinct molecular weights which could be the products of the family oftranscripts that we have observed.

Previously, we and others (See above and Gerster, T. et al. EMBO J.6:1323-1330 (1987); Landolfi et al., Nature 323:548-51 (1986)) showedthat the octamer binding protein NF-A2 varied considerably in expressionamong lymphoid cell lines. We therefore investigated the relationshipbetween levels of expression of the OCT-2 gene and levels of NF-A2 asjudged by mobility shift analysis. BJAB, the cell line which expressedthe largest amount of transcript showed the largest amount of NF-A2.Nuclear extracts from the pre-B cell lines, 38B9 and 70Z, showed verylittle NF-A2 and, correspondingly, expressed very little transcript(more poly(A)-containing mRNA from these two cell lines was loaded tosee a readily detectable signal). Of the three T lymphoma cell linestested, Jurkat, HUT78 and EL4, EL4 was the only line that showed largeamounts of NF-A2. Although NF-A2 was previously believed to be expressedonly in lymphoid cells we found that nuclear extracts from the gliomacell line that expressed the OC-2 gene contained an octamer bindingprotein which comigrated with NF-A2 in the mobility shift assay. Nuclearextracts from two glioma cell lines which were negative for OCT-2expression did not contain NF-A2. We have at present no explanation forthis apparent non-lymphoid expression of NF-A2 and the cloned octamerbinding protein gene. Previously, we had shown that NF-A2 but not NF-A1was inducible in pre-B cells by treatment of the cells with bacteriallipopolysaccharide (LPS) and that this induction required new proteinsynthesis. Therefore, we prepared poly(A)-containing mRNA from the pre-Bcell line 70Z/3 before and after LPS treatment and observed that LPSincreased the expression of the OCT-2 gene. Thus, in every instance, theexpression of the OCT-2 gene correlated with the presence of NF-A2 andis thus a good candidate for the gene which encodes NF-A2.

Further evidence that the oct-2 gene encodes NF-A2 was discovered whenthe NF-A2 factor was purified from nuclear extracts of BJAB cells byconventional chromatography followed by multiple passages over anaffinity column containing immobilized oligomers of the octanucleotidesequence. The purified NF-A2 consisted of three bands, as resolved bygel electrophoresis: a major band and two minor bands with deducedmolecular weights of 61 kD and 58 kD, and 63 kD, respectively. A cDNA(pass-3) for the oct-2 gene was inserted into the polylinker of the pGEM(Promega) expression vector. Translation of RNA transcribed from the SP6promoter-pass-3 cDNA construct yielded a major polypeptide from thepurified sample of NF-A2.

The mobility of a DNA-protein complex in the gel assay is primarilydetermined by the molecular weight of the protein. Complexes weregenerated with the affinity purified NF-A2 and the products oftranslationa in vitro of RNA from the oct-2 cDNA. These complexesco-migrated during electrophoresis in a native gel, again suggestingthat the oct-2 cDNA encodes the major form of the NF-A2 factor.

The affinity purified NF-A2 protein and the polypeptide translated invitro from the oct-2 cDNA were also compared by partial trypticdigestion. Samples from different digestion times of NF-A2 were resolvedby denaturing gel electrophoresis and detected by staining with silver.The mobility of these partial fragments was compared with those observedafter a parallel analysis of ³⁵ S-methionine labelled polypeitide fromtranscription/translation of the pass-3 CDNA in vitro. The two samplesgenerated a similar set of digestion fragments, again suggesting thatNF-A2 is encoded by the oct-2 cDNA.

Protein sequence comparisons suggested that the DNA binding domain ofoct-2 was specified by a domain (positions 952-1135) that was distantlyrelated to both the helix-turn-helix structure of bacterial repressorsand the homeobox-proteins. To directly test this analogy a fragment ofthe CDNA encompassing this region (655 to 1710) was inserted into theexpression vector pBS-ATG so that RNA could be transcribed from thetruncated templates by bacteriophage T7 RNA polymerase as indicated inFIG. 19A. The polypeptides translated in vitro from these RNAs weretested for specific DNA binding by addition of the total translation mixto the DNA-protein gel assay. Polypeptides produced from RNAsterminating at postions 1710 (Kpnl), 1443 (Stul), and 1134 (Pstl)specifically bound the octanucleotide containing probe, while thepolypeptide translated from RNA terminating at the 945 (Eagl) site didnot specifically bind. The region containing the helix-turn-helixportion of oct-2 is deleted in the latter protein. Since the truncatedpolypeptide encoded by RNA from the latter template was efficientlytranslated in the reticulocyte reaction, this suggests that the specificbinding of the oct-2-protein requires the helix-turn-helix structure.

Two distinct but similarly migrating protein-DNA complexes were detectedin the sample generated by translation of RNA from the Stul cleavedtemplate. Faint slower migrating complex comigrated with the complexgenerated with templates cleaved by Kpnl. The presence of the twocomplexes in the Stul-sample is due to a partial digestion of theplasmid DNA. The slower migrating complex is probably produced byprotein terminated at the stop codon TAA located at position 1465. Thefaster migrating complex probalby reults from molecules terminated atthe Stul site. This interpretaton was supported by the resolution of two³⁵ S-labeled polypeptides during gel electrophoresis of the Stul sampleand confirms the positon of the temrination codon of oct-2.

Many sequence-specific binding proteins have an oligomeric structure.For example, bacterial repressor proteins typically bind sites withtwo-fold rotational symmetry by forming a similarly symmetric dimer(Ptashne, Cell Press and Blackwell Scientific Publications, (1986)). Itshould be noted that the binding site sequence of the oct-2 protein isnot symmetric but oligomeric proteins could bind to non-symmetric sites.Other examples of oligomerization of sequence-specific bindingprotiensare the GCN4 protein of yeast (Hope and Struhl, EMBO J. 6: 2781-2784,(1987)) and the C/EBP protein of mammals. In the latter case, anα-helical region with an amphipathic character reflected in the spacingof four leucine residues by exactly seven residues is thought to beresponsible for dimer formation (Landschultz et al., Science 240:1759-1764 (1988); Landschultz et al., Genes & Development 2: 786-800(1988)). A convenient assay for detection of dimerization ofsequence-specific bindignproteins is to co-translate RNAs encoding twodifferent size forms of the protein and test whether protein-DNAcomplexes with novel mobilities are generated (Hope and Struhl, EMBO J.6: 2781-2784 (1987). If only monomers bind to the probe, the samplecontaining the co-translated polypeptides will generated only thecomplexes detected when either RNA is assayed singularly. This was thecase with combinations of different length RNAs transcribed from theoct-2 cDNA segment. Specifically, cotranslationof RNAs from templatescleaved at Stul (1443) and Pstl (1134) did not generate novel bands inthe gel mobility assay. Thus, on the basis of this negative evidence, wesuggest that a single molecule of the oct-2 protein is present in theresolved DNA-protein complexes and that it does not require dimerizationfor binding to DNA.

Anti-sera raised in rabbits against a bacterial fusion proteincontaining oct-2 encoded sequence (prepared employing the vestor pRIT2T(Pharmacia)) recognized the native oct-2 protein in metabolicallylabeled (³⁵ S-methionine) human B cells.

The molecular cloning of a lymphoid-restricted octamer binding proteingene demonstrates that higher eukaryotes have adopted a strategy ofgenetic diversification of transcriptional regulatory proteins whichbind a common regulatory motif. The ubiquitous and lymphoid-specificoctamer binding proteins have indistinguishable DNA binding sites, yetappear to have distinct functional properties (Staudt, L. M. et al,Nature 323:640-643 (1986)). Structure-function analysis of cloned yeasttranscription factors (Petkovich, M. et al., Cell 330:444-450 (1987);Giguere, V. et al., Nature 330:625-629 (1987)) and steroid receptorrelated transcription regulatory activity of a transcription factoroften reside in discrete protein domains that can be experimentallyinterchanged. The present findings suggest that similar diversificationof function among proteins which bind the octamer motif has occurredduring evolution. The octamer motif has been shown to be necessary andsufficient for lymphoid-specific promoter activity (Fletcher, C. et al.,Cell 51:773-781 (1987)) and NF-A2 has been shown to function as atranscription factor using octamer containing templates in vitro(Scheidereit, C. et al., Cell 51:783-793 (1987)). A furtherunderstanding of the lymphoid-specific activity of immunoglobulinpromoters may now come from an understanding of the mechanismsunderlying the lymphoid-specific expression of the OCT-2 gene.

EXAMPLE 8

Induction of NF-KB in Cells in Which It is Not Constitutively Present

The following work demonstrates that NF-KB is inducible in cells otherthan B (lymphoid) cells. As described below, it has now been shown thatNF-KB is inducible in pre-B cells and in non-lymphoid cells. Inparticular, the following work demonstrates that: 1) NF-kB factor can beinduced by the mitogen lipopolysaccharide (LPS) in two cell linesrepresenting a pre-B stage of B cell differentiation; 2) induction ofthis factor involves a post-translational modification of a pre-existingprotein because the induction takes place even in the presence oftranslational inhibitors like cycloheximide and anisomycin; 3) thesetranslational inhibitors by themselves can at least partially induceNF-kB and synergize with LPS to produce a superinduction; 4) an activephorbol ester like PMA can induce NF-kB by itself, and the time-courseof this activation is more rapid than that with LPS alone; and 5) it isalso possible to induce this factor in cell lines other than thosehaving a pre-B phenotype by means of an appropriate stimulus (e.g., inthe human T cell line, Jurkat, by PHA and/or PMA or in HeLa cells withPMA). Thus, B cells and plasma cells appear to support constitutivepresence of this factor whereas in other cell types it can be inducedtransiently by an appropriate stimulus.

Experimental Procedures

Cell lines and Extracts: 70Z/3 and PD cells were grown in RPMI 1640medium supplemented with 10% inactivated fetal calf serum, 50 μMβ-mercaptoethanol and penicillin and streptomycin sulfate (pen-strep)antibiotics. LPS (GIBCO) stimulation was carried out with 10-15 μg/ml.For experiments using protein synthesis inhibitors and LPS, cellcultures were treated with inhibitors approximately 20 min prior toaddition of LPS. Cycloheximide (Sigma) was used to 10 μg/mo which causesgreater than 95% inhibition of protein synthesis in 70Z/3 cells (Wall,R., et al., Proc. Natl. Acad. Sci. USA 83:295-298, (1986). Anisomycin(Sigma) was used at 10 which causes approximately 99% inhibition ofprotein synthesis in HeLa cells (Groliman, A. P., J. Biol. Chem.242:3226-3233, 1967). Phorbol ester activation of 70Z/3 cells wascarried out using the active ester phorbol 12-myristate-13-acetate (PMA)or the inactive ester phorbol 12,13-didecanoate at a concentration of 25ng/ml for the times indicated in the text. All treatments were carriedout at cell densities varying between 5×10⁵ -10⁶ cells/ml. Jurkat cellswere grown in RPMI 1640 medium with 10% inactivated fetal calf serum andpen-strep antibiotics. Phytohemmagglutinin (PHA) treatment was done at 5μg/ml and PMA treatment at 50 μg/ml. HeLa cells were grown in MEM mediumwith 5% horse serum and pen-strep antibiotics. Phorbol ester (PMAtreatment was at 50 μg/ml with cell density varying between 7×10⁵ -10⁶cells/ml.

Nuclear extracts were generated essentially according to the protocol ofDignam, J. D. et al., Nucl. Acids Res. 11:1475-1489 (1983) and proteinconcentration were determined using a Bradford assay with serum albuminstandards.

Gel Binding Analysis: Gel binding analyses were carried out as describedearlier using a radioactive DdeI to HaeIII fragment (k3) derived fromthe enhancer (Sen and Baltimore, 1986). Levels of NF-KB induced byvarious stimuli were normalized to total protein present in theextracts. Further, analysis with a different fragment that contains abinding site for the ubiquitous factor NF-A, shows that this nuclearprotein remains at approximately constant levels in all of the extractsreported here. Thus, the modulation of NF-KB activity is not areflection of variability of nuclear factors in general under theseconditions. For competition experiments, the specific and non-specificcompetitor DNA's were included in the mixture prior to addition of theprotein. The competitor fragments μ300, μ400, KE and SV40E which havebeen described earlier (Sen and Balitmore, 1986) were isolated from lowmelting point agarose gels and quantitated by spotting onto ethidiumbromide-containing agarose plates.

NF-KB can be induced in pre-B cell lines with bacteriallipopolysaccharide

To examine whether NF-KB might be inducible in 70Z/3 cells, cells werestimulated with LPS for 20 hr and nuclear extracts derived from thesecells were assayed for the presence of NF-KB using the electrophoreticmobility shift assay described previously. Singh, H. et al., Nature,319:154-158 (1986). U.S. patent application Ser. No. 817,441, To assayfor NF-KB, a DNA fragment containing its binding site (K3 fragment; Senand Baltimore, supra,) was end-labelled and incubated with extractsderived from either unstimulated 70Z/3 (cells or LPS-stimulated 70Z/3cells in the presence of increasing amounts of the carrier poly d(IC).Binding reactions were carried out for 15-30 minutes at room temperaturein a final volume of 15 μl containing 9 μg of total protein 3.5 μg(lanes 2,4) or 4.5 μg (lanes 3,5) of nonspecific carrier DNA poly d(IC)and 0.2-0.5 μg of probe. Reaction products were fractionated byelectrophoresis through low ionic strength polyacrylamide gels andvisualized by autoradiography. One lane contained free DNA fragments;another lane contained nucleoprotein complex generated by interaction ofNF KB with the fragment 3 in a nuclear extract derived from the B cellline WEHI 231.

Unstimulated 70Z/3 cell extracts lacked a major band evident with B cellextracts. This nucleoprotein complex band was induced in the 70Z/3 cellsafter LPS treatment for 20 hr. The band was not competed away even with4.5 ugm of poly d(IC). This induction phenomenon was not restricted tothe 70Z/3 cell line; another pre-B cell line, PD (Lewis S., et al., Cell30:807-816 (1982)), was weakly positive for the factor prior toinduction; Sen and Baltimore, 1986) but was strongly induced by LPS. Anumber of other minor bands could be seen in the binding assay, some ofwhich were inducible and others not. The major inducible band comigratedwith the major band produced by B cell and plasma cell extracts(typified by WEHI 231 extracts in. We have earlier characterized thisband by competition experiments and localized the binding site of thefactor by methylation interference experiments defining the band as oneproduced by interaction of the NF-KB factor with the B site within theenhancer (a site containing the sequence GGGGACTTTCC). Thus two pre-Bcell lines, one with a rearranged K gene (70Z/3) and the other in theprocess of undergoing rearrangement (PD), are clearly inducible by LPSfor NF-KB activity.

Induction of NF-KB by LPS does not require protein synthesis

Recently it has been reported that induction of transcription in 70Z/3does not require new protein synthesis. Nelson, K. J. et al., Proc. Nat.Acad. Sci. U.S.A. 82:5305-5309 (1985). Thus, induction of geneexpression was evident in cells pretreated (10 min) with the translationinhibitors cycloheximide or anisomysin followed by stimulation with LPS.Further, Wall et al. reported that expression could be induced in thepresence of cycloheximide alone which led them to argue in favor of alabile repressor blocking the activation of genes in this cell line. SeeWall, R. et al. Proc. Nat. Acad. Sci. USA 83:295-298 (1986). Todetermine if these characteristics of transcriptional activation wereparalleled by changes in the levels of NF-KB, we analyzed extractsderived from 70Z/3 cells which had been treated either with LPS alone,or with a translation inhibitor alone or with both together. To be ableto make direct correlations with the published reports concerning theeffects of translational inhibitors on expression in pre-B cells, weexamined a 4 hr time point in these experiments, although maximalstimulation of expression by LPS takes 14-20 hr. Binding reactions werecarried out as detailed above legend and contained 2.5, 3.5 or 4.5 μgpoly dIG with each set of extracts. End-labelled K3 fragment was theprobe in lane 1) and was incubated with 9-11 μg or protein from extractsderived from: untreated 70Z/3 cells (lanes 2,3,4), 70Z/3 cells treatedfor 4 hr with 10 μg/ml of LPS (lanes 5,6,7), 70Z/3 cells treated for 4hr with 10 μg/ml of LPS and 10 μg/ml cycloheximide (lanes 8,9,10); 70Z/3cells treated with 10 μg/ml of cyclheximide alone (lanes 11,12,13) andWEHI 231 cells (lane 14). In accord with the transcriptional analyses,uninduced 70Z/3 cells were negative for NF-KB, and treatment with eitherLPS alone or with cycloheximide alone for 4 hrs induced the factor.Unexpectedly, stimulation of 70Z/3 with LPS in the presence ofcycloheximide for 4 hr gave a superinduction of NF-KB, increasing it toa level above that seen after a 20 hr induction.

Qualitatively, the same result was observed when anisomycin was used asa translation inhibitor. Binding reactions were as detailed above using2.5 and 3.5 μg of poly d(IC) and protein from untreated 70Z/3 cells(lanes 2,3); 70Z/3 cells after induction with LPS alone (lanes 4,5);70Z/3 cells with LPS induction in the presence of anisomycin (lanes6,7); 70Z/3 cells treated with anisomycin by itself (lanes 8,9) and theB cell WEHI 231 as a positive control (lane 10). The characteristicnucleoprotein complex is indicated by the arrow. Thus, the presence ofanisomycin (10 uM) during a 4 hr stimulation with LPS gave asuperinduction of NF-KB relative to either LPS alone or anisomycinalone. Once again, prior to LPS treatment there was no detectable NF-KBactivity in 70Z/3. Although treatment of 70Z/3 with cycloheximide aloneor with LPS alone gave approximately equivalent amounts of NF-KB, thelevel of NF-KB induced with anisomycin alone appeared to be much less.This is probably due to drug toxicity because, even after a shortexposure to anisomycin, the cells looked quite unhealthy. Presumablythis also accounts for lesser degree of superinduction seen with LPS andanisomycin. Thus the enhancer binding factor NF-KB appears to beinducible in 70Z/3 cells in the absence of protein synthesis. Further,it appears to be inducible by either of 2 different translationinhibitors alone and is superinduced when the cells are stimulated withLPS and the inhibitor.

Phorbol ester can induce NF-KB in 70Z/3

The tumor promoting phorbol ester, phorbol 12-myristate-13-acetate(PMA), has been shown to induce surface immunoglobulin in 70Z/3,presumably via activation of K transcription and transport of completeimmunoglobulin to the cell surface (Rosoff P. M. et al., J. Biol. Chem.259:7056-7060 1984; Rosoff, P. M. and Cantley, L. C., J. Biol. Chem. 2609209-9215, (1985). To determine if this activation is reflected in anincrease of NF-KB, we analyzed extracts derived from 70Z/3 cells after a4 hr stimulation with PMA at 50 ng/ml. Binding reactions using K3 as aproblem (lane 1) were carried out as detailed above with protein fromuntreated 70Z/3 cells (lane 2) or 70Z/3 cells that had been treated withPMA at 50 ng/ml for 4 hr (lanes 3,4). Lane 5 was the positive controlfor NF-KB in extracts from WEHI 231. There was a striking induction ofNF-KB activity in these extracts. Thus an active phorbol ester by itselfis capable of inducing NF- B activity in 70Z/3 cells, implicatingprotein kinase C as a possible intermediate in the post-translationalmodification reaction that produces NF-B in these cells (Bell, R. M.Cell 45:631-632 (1986); Nishizuka, Y. Nature 308:693-697, (1984)!. Aninactive phorbol ester (phorbol 12, 13 didecanoate) did not causeinduction of NF-KB under similar conditions.

Time course of activation of NF-B by LPS and PMA are different

LPS-mediated stimulation of surface Ig expression of mRNA accumulationreaches a maximum after at least one cell cycle, i.e., in 14-18 hr.Recent work has shown that LPS stimulation of RNA synthesis, as measuredby nuclear run on assays Nelson, K. J., et al., Proc. Natl. Acad. SciUSA 82:5305-5309 (1985); Wall et al. supra, (1986)! can be seen as earlyas 4 hr after stimulation and that the DNAse I hypersensitive siteassociated with the enhancer can be detected as early as 1 hrpost-stimulation. To examine the time-course of NF-KB induction, wegenerated 70Z/3 cell extracts after stimulation either by LPS or PMA forvarying lengths of time. Analysis for NF-KB activity using the bindingassay showed that the time course of activation of NF-KB by these twoagents was quite different. Binding reactions were carried out withextracts derived from 70Z/3 cells that had been treated with LPS at 10μg/ml (lanes 3-7) or PMA at 25 ng/ml (lanes 8-12) for various lengths oftime as shown above each lane in the figure. Lane 2 was a positivecontrol for NF-KB in WEHI 231 extracts. With LPS alone, a nucleoproteincomplex band reflecting the presence of NF-KB increased until 2 hrpost-stimulation after which a slight decrease occurred and then thelevel remained constant. By contrast, in PMA-stimulated cells, NF-KB wasdetected at maximal levels within 0.5 hr after stimulation, remained atthis level for 2-3 hours and then began to drop off rapidly, such thatby 8 hr it was barely detectable. Because prolonged exposure of cells tophorbol esters is known to result in desensitization of endogenousprotein kinase C (Rodriquez-Pena, A. and Rozengurt, E., Biochem Biophys.Res. Comm. 120:1053-1009, 1984; EMBO J, 5:77-83 1986), a possibleexplanation for the rapid decline of NF-KB may be that its maintenanceas a binding factor requires continuous activity of protein kinase C. Asimilar phenomenon has been described recently by Blemis and Eriksonwhere S6 kinase activity assayed by phosphorylation of S6 protein) firstrises and then falls during prolonged exposure to PMA. See Blemis, J.and Erikson, R. L. Proc. Natl. Acad. Sci. USA 83:1733-1737 (1986).Although it has been reported that LPS may directly activate proteinkinase C (wightman, P. D. and Raetz, C. R. H., J. Biol. Chem.259:10048-10052, 1984) the different kinetics of induction of NF-KB byLPS and PMA implies that these activators feed into a common pathwaythrough distinguishable sites of activation.

Non pre-B cell lines can also be activated to produce NF-KB

In our previous analysis we have shown that NF-KB is present only incell lines representing the B cell or plasma cell stages of B lymphoiddifferentiation, but was undetectable in a variety of non B cells, pre-Bcells and T cells (Sen and Baltimore, 1986). However, as shown above,this factor may be induced to high levels in pre-B cells uponstimulation with LPS. To check if this inducibility was restricted tocells having a pre-B phenotype only or was a general characteristic ofthe other constitutively negative cell lines we have takenrepresentative examples of cell types (T cells and non lymphoid cells)and examined then for induction of NF-KB after appropriate stimulation.

The human T leukemia cell line, Jurkat, can be stimulated to produceinterleukin-2 (IL-2) by the combined influence of phytohemagglutinin(PHA) and phorbol ester (PMA) (Gillis, S. and Watson, J., J. Alp. Med.,152:1709-1719, 1980; Weiss et al., J. Immunol. 133:123-128, 1984).Nuclear extracts were prepared from Jurkat cells that had beenstimulated with either PHA alone or PMA alone or both together andanalyzed for the presence of NF-KB. The human T lymphoma Jurkat wasstimulated with phytohemagglutinin (PHA) and phorbol12-myristate-13-acetate (PMA) individually or together for 20 hr.Nuclear extracts made after treatment were analyzed by the mobilityshift assay using K-3 fragment as the labelled probe. Binding reactionstypically contained 6 g of protein, 2.5-3.5 g of poly d(IC) and 0.3-0.5ng of end-labelled DNA probe. Lane 1: no protein added; lane 2: WEHI 231extract (positive control); lane 3: extract from uninduced Jurkat cells:lane 4: Jurkat cells stimulated with PHA alone; lane 5: Jurkat cellsstimulated with pHA and PMA; Lane 6: Jurkat cells stimulated with PMAalone. The arrow shows the position of the expected nucleoproteincomplex generated by interaction of NF-KB with K-3 fragment. Asoriginally observed, extracts derived from uninduced Jurkat cells werenegative for NF-KB activity. However, extracts made from Jurkat cellswhich had been stimulated either with PHA or PMA contained detectablelevels of NF-KB and the extracts from the co-stimulated cells showedhigher levels of the factor. Thus a factor with the properties of NF-KBcan be induced in a T cell lines after appropriate activation.

As an example of a non-lymphoid line we used the human HeLa cell lineswhich is constitutively negative for NF-KB (Sen and Baltimore, 1986).These cells were induced with PMA for 2 hr and extracts derived fromtreated and untreated cells were analyzed for NF-KB activity. HeLa cellswere treated with PMA (50 ng/ml) for 2 hrs. and the extracts derivedthereafter were analyzed for induction of NF-KB. Binding reactionscontained 15-18 μg of protein, 3.5 μg of poly d(IC) and 0.3-0.5 μg ofend-labelled DNA probe. Lane 1: 3 fragment/no protein added; lane 2: 3fragment incubated with extracts derived from the human B lymphoma EW;lane 3: K3 fragment incubated with uninduced HeLa cell nuclear extract;lane 4: μ50 fragment (derived from the μ-heavy chain enhancer andcontaining a copy of the conserved octamer sequence ATTTGCAT) incubatedwith uninduced HeLa cell extracts; lane 5: K3 fragment incubated withinduced HeLa cell extracts. The untreated HeLa extract did not show anucleoprotein complex which comigrated with the complex generated in Bcell extracts. However treatment with PMA induced a factor thatgenerated the characteristic DNA-protein complex produced by NF-KB. As acontrol, both the uninduced and induced extracts showed equivalentlevels of the ubiquitous NF-A1 DNA binding protein when analyzed using aprobe containing the sequence ATTTGCAT (Singh, H. et al., Nature319:154-158, 1986). Therefore treatment of HeLa cells with PMA induces afactor that can form a nucleoprotein complex with the K3 fragment.

To further characterize the DNA-protein complex formed in thePMA-treated HeLa cell extracts, we carried out competition experiments.Binding reactions were carried out using end-labelled K3 fragment, 3.5μg of poly d(IC) and 15-18 μg of nuclear extract in the present of 50 ngof unlabelled competitor DNA derived from various immunoglobulin andviral regulatory sequences. The complex generated in PMA-induced HeLacell extracts was specifically competed away by the inclusion of 50 ngof unlabelled DNA in the binding reaction containing either the enhancedor the SV40 enhanced but was unaffected by two DNA fragments thattogether span the K enhancer, or by a 250 bp fragment containing the Kpromoter. This pattern of competition exactly parallels the patternobserved earlier using the K3 fragment in binding experiments with Bcell derived extracts (Sen and Baltimore, 1986). These results furtherstrengthen the conclusion that the NF-KB factor can be induced innon-lymphoid cells as well as lymphoid cells following appropriatestimulation.

EXAMPLE 9 Characterization of the NF-kB Protein and ElectrophoreticMobility Shift Analysis of Subcellular Fractions of 70Z/3 Cells

Characterization of the NF-kB protein

Mouse NF-kB is a polypeptide with a molecular weight around 60 kDa. Thishas been determined by a DNA-binding renaturation experiment usingeluates from different molecular weight fractions of a reducing SDS-gel(FIG. 15A). The size of the native lNF-kB protein was determined in thefollowing manner: Nuclear extract from TPA-stimulated cells wassubjected to ultracentrifugation on a continuous glycerol gradient. Thefractions were assayed for DNA-binding activity of NF-kB byelectrophoretic mobility shift assays (FIG. 15B). NF-kB activity wasfound highest between the co-sedimented bovine serum albumin (67 kDa)and IgG (158 kDa) standards (FIG. 15B, lanes 6 to 8). The specificity ofbinding was shown by the absence of a complex when a DNA probe with amutation in the NF-kB binding sequence was used to assay the fractions(FIG. 15B, right lanes 4 to 10). Lenardo, M. et al., Science,236:1573-1577 (1987). Little NF-kB activity was contained in thefractions where a 60 kDa protein would be expected to sediment (FIG.15B, lanes 4 and 5). It the sedimentation of NF-kB is not highlyabnormal, the results from the glycerol gradient centrifugation suggestthat NF-kB is associated with another protein of approximately the samesize. Presumably NF-kB forms a homodimer because the protein-DNA complexformed in native gels using whole nuclear extract is of the samemobility as the complex formed with renatured NF-kB protein from asingle spot of a two-dimensional gel.

70Z/3 cell cultures were incubated in the absence (Co) and presence ofphorbol ester (TPA), followed by subcellular fractionation of cells. Inthe DNA-binding reactions, 8.8 ug of protein of nuclear extracts (N),cytosolic fractions (C), and post-nuclear membrane fractions (P) in 4 ulbuffer D(+) were used. The end-labeled DNA-fragments were incubated inthe presence of 3.2 ug poly(d I-C!) with the subcellular fractions in afinal volume of 20 ul for 15 to 30 minutes followed by separation ofprotein-DNA complexes and unbound DNA on native 4% polyacrylamide gels.Fluorograms of native gels are shown. To detect kB-specific DNA-bindingactivity a DdeI-HaeIII wild type fragment of the kappa light chainenhancer (kB wt; lanes 1-6) was used. Sen, R. and D. Baltimore, Cell,46:705-716 (1986). kB-un-specific activities binding to the kappaenhancer fragment were detected using a fragment mutated in NF-kBbinding site that was otherwise identical to the wild type fragment(lanes 7-12). Lenardo, M. et al., Science, 236:1573-1577 (1987).NF-uE3-binding activity and octamer binding protein activity wereassayed with a HaeIII-DdeI kappa enhancer fragment (uE3; lanes 13-18)and a PvuII-EcoRI kappa heavy chain promoter fragment (OCTA; lanes19-24), respectively. Sen, R. and D. Baltimore, Cell, 46:705-716 (1986);Singh, H. et al., Nature, 319:154-158 (1986). Specific protein-DNAcomplexes are indicated by filled arrowheads and the positions ofunbound DNA-fragments by open arrowheads.

EXAMPLE 10 Renaturation of NF-kB

70Z/3 cells were grown in spinner cultures with RPMI 1640 mediumsupplemented with 10% newborn calf serum and 50 uM 2-mercaptoethanol.HeLa cells were also grown in spinner cultures with MEM mediumsupplemented with 10% horse serum. Cell cultures were treated with 25ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA; Sigma) for 30 minutesat cell densities between 7×10⁵ and 2×10⁶ /ml.

Subcellular Fractionation

Cells were collected by centrifugation for 10 minutes at 150×g. Cellpellets were resuspended in ice-cold phosphate-buffered saline andcollected again by centrifugation. All following steps were carried outat 4° C. Washed cells were resuspended in four packed cell volumes of ahypotonic lysis buffer (buffer A; Dignam, J. P. et al., Nucleic AcidResearch, 11:1475-1489 (1983)). After 20 minutes, cells were homogenizedby 15 (HeLa) or 20 strokes (70Z/3 cells) with a loose fitting Douncehomogenizer. Nuclei were collected by centrifugation for 6 minutes at4300×g, resuspended in five volumes of buffer A and washed once bycentrifugation. Proteins were extracted from washed nuclei by high salt,followed by centrifugation of the nuclear extracts and dialysis againstbuffer D as described. Dignam, J. P. et al., Nucleic Acids Research,11:1475-1489 (1983). One percent NP-40 (v/v) was added to the dialyzednuclear extracts. The postnuclear supernatant was centrifuged for 6minutes at 4300×g and the resulting supernatant ultracentrifuged for 1hour at 150,000×g. The pellet after ultracentrifugation containingpostnuclear membranes was dissolved in buffer D containing 1% (v/v)NP-40 (referred to as buffer D(+)). Insoluble material was removed bycentrifugation for 10 minutes in a Microfuge. The supernatant afterultracentrifugation (referred to as cytosolic fraction) was adjusted tobuffer D(+) conditions by the addition of stock solutions. Fractionswere stored at -70° C.

Protein concentrations were determined by an assay using bicinchoninicacid. Smith, P. K. et al., Anals of Biochemistry, 150:76-85 (1985). Theratio of the total protein recovered during a fractionation experimentin nuclear extracts, cytosolic fractions and postnuclear membranefractions was 2:4:1 for 70Z/3 cells and 1:10:1.5 for HeLa cells. Theseratios were used to adjust the fractions to protein concentrationsreflecting equal cell-equivalents of subcellular fractions.

Electrophoretic Mobility Shift Assays and Treatments with DissociatingAgents

DNA-binding reactions were carried out as described above. TheDNA-binding reaction mixture contained poly(d I-C!) (Pharmacia),3000-6000 cpm of ³² P!end-labeled DNA-fragments and a buffer composed of10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM EDTAand 5% glycerol. Binding reactions and subsequent analysis on native 4%polyacrylamide gels were performed at room temperature as described.Sen, R. and D. Baltimore, Cell, 46:705-716 (1986). Subcellular fractionswere treated with formamide (DNA grade; American Bioanalytical) prior tothe addition of the DNA-binding reaction mixture. Sodium deoxycholate(Fisher Scientific Company) was added after the DNA-binding reaction.

Renaturation of NF-kB

Protein in the subcellular fractions was precipitated at -20° C. by theaddition of four volumes of acetone. Pellets were dissolved inSDS-sample buffer containing 3.3% 2-mercaptoethanol and boiled for 5minutes. Laemmli, U. K., Nature, 227:680-685 (1970). AfterSDS-polyacrylamide gel electrophoresis, gel pieces from differentmolecular weight regions were cut out, ground, and proteins elutedovernight at 4° C. in 500 ul of a buffer containing 50 mM Tris-HCl, pH7.9, 0.1% SDS, 0.1 mg/ml bovine serum albumin, 1 mM DTT, 0.2 mM EDTA,0.1 mM phenylmethylsulfonyl fluoride (PMSF) and 2.5% glycerol. Aftercentrifugation for 2 minutes in a Microfuge, the supernatant was removedand recentrifuged for 10 minutes to remove gel debris.

To 200 ul of the supernatant four volumes of acetone were added andproteins were allowed to precipitate for 2 hours at -20° C. Theprecipitate was collected by centrifugation for 10 minutes in aMicrofuge, washed once with 1 ml methanol at -20° C. and dried for 30minutes in the inverted tube. The dried pellet was dissolved in 2.5 ulof a saturated solution of urea (ultrapure; American Bioanalytical) anddilued with 125 ul of a buffer containing 20 mM Tris-HCl, pH 7.6, 10 mMKCl, 2 mM DTT and 10 uM PMSF. Renaturation was allowed for a minimum of18 hours at 4° C. kB-specific DNA-binding activity was detectable inmobility shift assays for at least 48 hours after storage of renaturedfractions at 4° C. without appreciable loss of activity.

EXAMPLE 11 Subcellular Localization of the NF-kB Precursor

Because NF-kB is a DNA-binding protein, it is expected to reside in thenucleus. This is certainly true for NF-kB of phorbol ester-treated cellsand mature B-cells where the activity is detectable in nuclear extracts.It is however not mandatory for a precursor of NF-kB especially, in viewof the fact that the precursor is activated by protein kinase C, acytosolic protein that is associated in its active state with the plasmamembrane.

The precursor of NF-kB was analyzed by an investigation of subcellularfractions of unstimulated pre-B cells for the presence of NF-kBactivity, using electrophoretic mobility shift assays. LittleDNA-binding activity was detected in the subcellular fractions,indicating that the precursor must exist in a form of low affinity forits cognate DNA. In fractions from TPA-stimulated cells, the newlyactivated NF-kB was almost exclusively contained in the nuclear extract.

In an attempt to activate the DNA-binding activity of the NF-kBprecursor, the various subcellular fractions were treated with agentsknown to gently dissociate protein-protein interactions. Lowconcentrations of desoxycholate, formamide or a combination of bothincluded in the mobility shift assay mixture led to the activation of anNF-kB-specific DNA-binding activity. The fraction containing the bulk ofthe in vitro activatable NF-kB precursor was the cytosol. When fractionsfrom TPA-stimulated cells were subjected to the same treatment, theamount of precursor in the cytosolic fraction was found stronglyreduced, apparently because of redistribution of activated NF-kB intothe nuclear extract fraction. In both control and TPA-stimulated cells,the amount of total cellular NF-kB activity revealed after treatmentwith dissociating agents was equal, suggesting a complete conversion ofthe NF-kB precursor into active NF-kB. Cytosolic fractions from HeLacells and from calf spleen also contained NF-kB precursor which could bedemonstrated after activation with dissociating agents. Theseobservations strongly suggest that NF-kB is localized as an inactiveprecursor in the cytosol. Activation of protein kinase C by phorbolester then would result in two events: induction of DNA-binding activityand nuclear translocation of NF-kB.

Using subcellular fractions from HeLa cells, another TPA-inducibletranscription factor, AP-1, was tested to determine whether it alsoexhibits activation and subcellular redistribution upon TPA-stimulation.As detected by mobility shift assays, AP-1 from nuclear extracts did notshow an increase in DNA-binding activity after TPA-stimulation nor werethere significant amounts of AP-1 activity present in the cytosolicfractions from control and stimulated cells. This showed that themechanism by which the transcription factor activity of NF-kB is inducedis fundamentally different from that of AP-1 although the initialsignal--activation of protein kinase C by phorbol ester--is the same.

EXAMPLE 12 Investigation of the DOC-dependence of Cytosolic NF-kB

Cytosol from unstimulated 70Z/3 pre-B cells in buffer A (Dignam, J. P.et al., Nucl. Acids. Res., 11:1475 (1983); Baeuerle, P. A. and D.Baltimore, Cell, 53:211 (1988)) was adjusted to a final concentration of50 mM NaCl, 20 mM (HEPES) (pH 7.9), 1.5 mM EDTA, 5% glycerol and 0.2%NP-40. Cytosolic protein (45 mg) was mixed to a final volume of 4 mlwith 0.6% DOC, 0.75 g calf thymus (wet weight) DNA-cellulose Sigma;equilibrated in buffer G: 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mMEDTA, 1 mM dithiothreitol (DTT), 5% glycerol, 0.2% DOC, 0.2% NP-40, and0.5 mM phenylmethyl sulfonylfluoride (PMSF)! and 1.2% NP-40. Thesuspension was incubated in a mini column for 1 hour at room temperatureon a rotary shaker. The flow-through fraction was used for gelfiltration. DNA-cellulose was washed with buffer G and eluted with aNaCl step gradient in buffer G. Equal proportions of fractions wereassayed by EMSA (Sen, R. and D. Baltimore, Cell, 46:705 (1986);Baeuerle, P. A. and D. Baltimore, Cell, 53:211 (1988)) at a finalconcentration of 1.2% NP-40 in the presence of either 0.03% DOC (nondissociating condition) or 0.6% DOC (dissociating condition) and with 10μg of bovine serum albumin (BSA) as carrier. Results of thisinvestigation are described above.

EXAMPLE 13 Characterization of IkB and its Complex with NF-kB

The flow-through fraction from the DNA-cellulose column (1.55 mg ofprotein in 250 μl described in Example 4) was subjected to a G-200Sephadex column (280 by 7 mm) with a flow rate of 0.15 ml/min in bufferG at room temperature. A mix of size markers (dextran blue;immunoglobulin G, 158 kDa, BSA, 67 kDa, ovalbumin, 45 kDa, myoglobin, 17kDa; Biorad) was run separately on the column prior to sample runs.Markers were detected in fractions by their color and usingSDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by CoomassieBlue staining.

To detect inhibiting activity, portions of fractions (5 μl; in buffer G)were mixed with 1 μl of nuclear extracts in buffer D(+)! and 0.5 μl 10%NP-40. Dignam, J. P. et al., Nucl. Acids Res., 11:1475 (1983). After 30minutes at room temperature, the reaction volume was brought to 20 μl bythe addition of a DNA binding reaction mixture containing 3.2 μg ofpoly(dI-dC) (Pharmacia), 5 to 20 fmoles of ³² P-end labeled k enhancerfragment, 75 mM NaCl, 15 mM Tris-HCl (pH 7.5), 1.5 mM EDTA, 1.5 mM DTT,7.5% glycerol, 0.3% NP-40 and 20 μg BSA. After a 20-minute DNA bindingreaction, samples were analyzed by EMSA.

Gel filtration fractions containing IkB (25 μg of protein) wereincubated for 1 hour at room temperature in buffer G without anyaddition or with 2 μg of TPCK-treated trypsin (Sigma), 8 μg of BPTI(Sigma), or with 2 μg of trypsin that had been incubated with 8 μg ofBPTI. Tryptic digestion was stopped by a 10-minute incubation with 8 μgof BPTI and samples analyzed as described above.

Nuclear extract from TPA-stimulated 70Z/3 cells and cytosol fromuntreated cells (both 220 μg of protein) were sedimented through 5 ml ofa continuous 10 to 30% glycerol gradient in buffer D(+) and 150,000 g(SW 50.1 rotor; Beckman) for 20 hours at 4° C. Cosedimented size markerswere detected in fractions by SDS-PAGE and Coomassie Blue staining.Portions of glycerol gradient fractions (4 μl) were analyzed by EMSAwith 10 μg of BSA as carrier and 0.5 μg of poly(dI-dC). NF-kB precursorwas activated by treating 4 μl of fractions with 1.5 μl of formamidebefore the DNA binding reaction mixture was added.

EXAMPLE 14 Demonstration of the Presence of the NF-kB--IkB Complex inEnucleated Cells

HeLa cells were grown in Eagle's Minimum Essential Medium supplementedwith 10% horse serum, penicillin (50 I.U./ml) and streptomycin (50μg/ml) (referred to as MEM-medium) on discs (1.8 cm in diameter) cutfrom cell culture plastic ware. For enucleation, discs were placedupside down into centrifuge tubes filled with 10 ml of MEM-medium of 37°C. containing cytochalasin B (10 μg/ml) and held for the same time inthe incubator. To estimate the enucleation efficiency, enucleated cellson one disc were fixed with formaldehyde (3.7%) in phosphate-bufferedsaline (PBS) for 20 minutes, stained for 4 minutes with4',6-diamidino-2-phenylindole (DAPI, 1 μg/ml; Sigma) in PBS, and washedin PBS. Fluorescence microscopy under UV light and phase contrastmicroscopy were performed with a Zeiss Photomicroscope III. Control andenucleated cells were allowed to recover in cyfor alasin B-freeMEM-medium for 30 minutes before a 2-hour incubation in the absence orpresence of TPA (50 ng/ml). Cells were then washed in ice-cold PBS,scraped off the discs in 100 μl of a buffer containing 20 mM HEPES (pH7.9), 0.35M NaCl, 20% glycerol, 1% NP-40, 1 mM MgCl₂, 1 mM DTT, 0.5 mMEDTA, 0.1 mM EGTA, 1% aprotinin (Sigma) and 1 mM PMSF. After lysis andextraction for 10 minutes on ice, particulate material was removed bycentrifugation (Microfuge) for 15 minutes at 4° C. and the resultingsupernatants were analyzed by EMSA.

EXAMPLE 15 Demonstration of the Role of NF-KB as Mediator in Regulationof a Gene in Non-Lymphoid Cells

The following demonstrates that NF-κB has the role of mediator incytokine gene regulation (in this case, positive regulation of β-IFNgene expression). NF-κB has been shown to interact with avirus-inducible element (PRDII) in the β-IFN gene and to be highlyinduced by either virus infection or treatment of cells withdouble-standard RNA.

A. Experimental Procedure

Cell Culture and Transfection

Mouse L929 fibroblasts were maintained in MEM medium (Gibco) with 5%serum (Gibco). Jurkat (human T lymphocytes), Namalwa (human Burkittlymphoma), S194 (mouse myeloma), and 70Z/3 (mouse pre-B lymphocyte)cells were grown in RPMI 1640 medium supplemented with 10% fetal calfserum (Life Science) and 50 μM β-mercaptoethanol. Sendai virus (SPAFAS)or poly(rI:rC) (Pharmacia) inductions were either 6 hours in length forprotein extracts (Zinn et al., Cell, 34:865-879 (1983)) or 12 hours fortransfections. Phorbol myristate acetate (Sigma) and phytohemagglutinin(PHA) induction was carried out as described by Sen, R. and D. Baltimore(Cell, 47:921-928 (1986)).

Transient transfections of L929 cells were performed according to Kuhlet al., Cell, 50:1057-1069 (1987); and for S194 cells, according toPierce et al., Proc. Natl. Acad. Sci. USA, 85:1482-1486 (1988). Aβ-galactosidse (β-gal) expression plasmid (Edlund et al., Science,230:912-916 (1985)) was co-transfected to monitor the transfectionefficiency. CAT assays were described by Gorman et al., Mol. Cell.Biol., 2:1044-1051 (1982), and the amount of protein assayed wasnormalized to a constant amount of β-gal activity. An et al., Mol. Cell.Biol., 2:1628-1632 (1982).

Plasmid constructions

The plasmids p-41βCAT (-41β) , p-41PII4r (-41β(P)₄), and p-41PII2r(-41β(P)₂) were constructed as follows: in Fan, C. M. and T. Maniatis,EMBO J., in press (1989). The nucleotide sequence of the PRDII×2(PRDII₂) is 5'-GATCTGTGGGAAATTCCGTGGGAAATTCCGGATC-3'. The constructionof Δ56 (c-fosCAT), Δ56(B)₂ (J16), and Δ56(B⁻) (J32) were described byPierce et al., Proc. Natl. Acad. Sci. USA, 85:1482-1486 (1988). The κBoligonucleotides were: Wild-type: 5'-TCGACAGAGGGGACTTTCCGAGAGGCTCGA-3'and mutant: 5'-TCGACAGAATTCACTTTCCAGGAGGCTCGA-3'. The IRE was isolatedas a BglII-BamHI fragment (Goodbourn et al., Cell, 45:601-610 (1986))and cloned into pSP73. Mutant PRDII sites were described in Goodbourn,S. and T. Maniatis, Proc. Natl. Acad. Sci. USA, 85:1447-1451 (1988).

Preparation of subcellular protein fractions and mobility shiftelectrophoresis

Buffers A, C and D are those described by Dignam et al., Nucl. AcidsRes., 11:1475-1489 (1983). Frozen pellets containing from 1×10⁶ to 1×10⁷cells were thawed in the presence of an equal volume of buffer A. Thesuspension was mixed using 10 strokes of a Dounce homogenizer and thenuclei were pelleted for 20 minutes at 4° in a microcentrifuge. Thesupernatant, termed cytosol, was ultracentrifuged for 1 hour at100,000×g and adjusted to 20% glycerol, 10 mM HEPES, pH 7.9, 1 mM EDTA,and 0.1M KCl. The nuclei were extracted with 2 volumes of the buffer Cfor 20 minutes and the nuclear extract was cleared by centrifugation anddialyzed against buffer D. To minimize proteolysis, all buffers included0.5 mM PMSF, 0.3 μg/ml leupeptin, and 0.3 μg/ml antipain and buffers Aand C included 0.3 TIU/ml aprotinin, 0.5 mg/ml benzamidine, 0.1 μg/mlchymostatin, and 0.7 μg/ml pepstatin.

Binding assays were carried out as described in Lenardo et al., Science,236:1573-1577 (1987) and Lenardo et al., Proc. Natl. Acad. Sci. USA,85:8825-8829 (1988). Assay samples of 20 μl contained nuclear extractincubated with 0.25 ng ³² P-labeled DNA fragment (5,000 CPM), 10 mMTris:Cl, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA, 0.5 mM MgCl₂, 3 mM GTP(omitted in experiments varying amounts of added GTP), 2 μg poly(dI-dC),and 5% glycerol for 20 minutes at room temperature. Cytosol wasactivated in vitro using 0.8% sodium deoxycholate followed by additionof the binding mixture including 0.75% NP-40. Methylation interferenceassays were performed using the procedure of Gilman et al., Mol. Cell.Biol., 6:4305-4316 (1986).

RNA analysis

RNA preparation and Northern blot analysis were carried out aspreviously described by Zinn et al., Cell, 34, 865-879 (1983).

B. Results

NF-κB binds specifically to PRDII

The protein encoded by the PRDII-BF1 cDNA binds to the PRDII site and tothe H₂ -K^(b) and κB binding sites (FIG. 17, Singh et al., Cell,52:415-423 (1988); Fan and Maniatis, unpublished). Thus, the ability ofNF-κB to bind to PRDII and to the κB site was compared, using anelectrophoretic mobility shift assay. Fried, M. and Crothers, D. M.,Nucleic Acid Res., 9:6505-6525 (1981). NF-κB is present in the human Tlymphocytic line Jurkat in an inactive form, but its binding isinducible by phorbol myristate acetate (PMA) and phytohemagglutinin(PHA). Sen, R. and D. Baltimore, Cell, 47:921-928 (1986); and Nabel, G.and D. Baltimore, Nature, 326:711-713 (1987). The entire interferon generegulatory element (IRE) or an oligonucleotide comprised of two copiesof the PRDII sequences (PRDII₂ or (P)₂) was used. A complex withPMA/PHA-induced Jurkat nuclear extracts that migrated identically tothat formed with the κB site was detected. It was assumed that theadditional slower migrating complex observed with the PRDII₂oligonucleotide corresponds to DNA molecules in which both of the PRDIIsites are bound to protein. Specific κB complexes were undetectable inextracts from unstimulated Jurkat cells.

To determine whether the same protein binds to PRDII and κB, competitionexperiments were carried out. Increasing amounts of unlabeled κBoligonucleotide inhibited complex formation with either the IRE or theκB site. The ability of PRDII and KB to compete for NF-κB binding wasalso reciprocal; an unlabeled fragment prevented complex formation witheither labeled fragment. Quantitatively, both sites competed equally forNF-κB. Furthermore, the IRE and the κB sites both formed an identicalcomplex using cytosol from unstimulated Jurkat cells after treatmentwith the detergent deoxycholate. This observation is in agreement withthe finding that NF-κB binding activity can be unmasked in cytosolicextracts by deoxycholate. Baeuerle, P. and D. Baltimore, Cell,53:211-217 (1988).

A number of single base mutations in PRDII decrease the level of virusinduction of the β-IFN gene. Goodbourn, S. and T. Maniatis, Proc. Natl.Acad. Sci. USA, 85:1447-1451 (1988). Therefore, these mutations wereexamined to assess whether they also affect in vitro binding to NF-κB.Four point mutations that impair inducibility of the β-IFN gene wereshown to reduce binding of NF-κB. (64G→A, 62G→A, 60A→G and 56C→T). Asingle point mutation that has no effect on inducibility allows specificNF-κB binding (65T→C). Taken together, these results strongly suggestthe NF-κB plays a direct role in β-IFN gene regulation.

The in vivo activities of PRDIII and the κB site are indistinguishable

To determine whether the PRDII and κB sites function similarly in vivo,we compared their transcriptional activities were compared, usingchloramphenicol acetyltransferase gene (CAT) reporter plasmids invirus-induced mouse L929 fibroblasts and in S194 mouse myeloma cells.The structures of the reporter genes are illustrated in FIG. 18B. The-41 β-globin/CAT gene was not expressed in L929 cells before or afterinduction by inactivated Sendai virus or poly(rI:rC) (FIG. 18A, lanes1-3). However, the reporter gene linked to four copies of PRDII(-41β(P)₄) was highly inducible (FIG. 18A, lanes 4-6). Remarkably, twotandemly-repeated κB sites also conferred virus and poly(rI:rC)inducibility on a c-fos promoter/CAT fusion gene in L929 cells (FIG.18A, compare lanes 7-9 to 10-12). Mutations in the κB site thateliminated binding of NF-κB also abolished virus inducibility. Thus, inL929 cells, the same inducible factor may interact with PRDII and the κBsite to stimulate transcription.

The B-cell specific activities of PRDII and the κB site were compared bytransfecting the reporter enes into S194 mouse myeloma cells. Aspreviously demonstrated, wild-type, but not mutant, κB sites were highlyactive in mature B-cells which constitutively express NF-κB; Pierce etal., Proc. Natl. Acad. Sci. USA, 85:1482-1486 (1988)). Significantly,multiple PRDII elements were also highly active in S194 cells (lanes16-18). These results further suggest that the same factor, NF-κB,interacts productively with PRDII and the κB site.

Virus induction stimulates NF-κB binding in lymphoid and non-lymphoidcells

The ability of PRDII to bind NF-κB suggested that virus infection mightactivate NF-κB. Therefore, nuclear extracts prepared from cells beforeand after virus induction were analyzed. NF-κB binding activity wasvirus-inducible in Namalwa human mature B lymphocytes, 70Z/3 murinepre-B lymphocytes, and murine L929 fibroblasts. Virus induction of NF-κBin Namalwa cells was unexpected because these cells display significantconstitutive NF-κB binding activity in the nucleus. Thus, only afraction of the NF-κB in Namalwa cells is in the active state, and theremaining molecules can be activated by virus. Virus-induced complexesin all three cell types appeared to contain NF-κB because they could beeliminated by competition with wild-type (WT) but not mutant (MUT) κBsites. Moreover, complex formation could be stimulated by GTP, abiochemical property of NF-κB. Lenardo et al., Proc. Natl. Acad. Sci.USA, 85:8825-8829 (1988). The NF-κB complex was also induced bypoly(rI:rC) in 70Z/3 cells, but the effect was less dramatic.

Additional evidence that the virus-induced complexes contain NF-κB wasprovided by comparisons of the methylation interference patterns of thevirus-induced complexes from Namalwa and L929 cells with thePMA/PHA-induced complex in Jurkat cells. All of the interferencepatterns were identical and exhibited the close base contacts that aredistinctive of the interaction between NF-κB and its cognate bindingsite in the κ enhancer. Sen, R. and D. Baltimore, Cell, 46:705-716(1986); and Baldwin, A. S. and P. A. Sharp, Mol. Cell. Biol., 7:305-313(1988). Finally, the level of NF-κB revealed by deoxycholate in thecytosol of L929 cells was diminished after virus induction. Therefore,like phorbol ester treatment, virus infection apparently releases NF-κBfrom an active cytosolic form and allows translocation to the nucleus.Baeuerle, P. and D. Baltimore, Cell, 53:211-217 (1988).

Endogenous β-IFN and Ig kappa gene expression are activated by virus inpre-B lymphocytes

As shown above, virus infection dramatically increased the levels ofnuclear NF-κB and induced reporter genes containing PRDII or κB sites.Therefore, the ability of virus to induce the transcription of anendogenous κ gene was also assessed. The pre-B cell line 70Z/3, whichproduces cytoplasmic Ig μ heavy chains, but not light chains, was usedfor this purpose. Paige et al., J. Immunol., 121:641-647 (1978). The κgene in 70Z/3 cells is functionally rearranged and can betranscriptionally induced by lipopolysaccharide (LPS) and phorbolmyristate acetate (PMA), conditions which powerfully induce NF-κB.Nelson et al., Nucl. Acids Res., 12: 1911-1923 (1984); Rosoff andCantley, J. Biol. Chem., 259:7056-7060 (1985); and Sen, R. and D.Baltimore, Cell, 47:921-928 (1986). As expected, treatment of 70Z/3cells with phorbol esters or, more strikingly, with LPS resulted in theactivation of the endogenous κ gene. Surprisingly, virus infection alsoinduced κ gene expression to a level comparable to that observed withPMA induction. Under the same conditions, endogenous β-IFN mRNA wasinduced by virus, but not by PMA or LPS. Thus, NF-κB is necessary andsufficient for expression of the endogenous κ gene in 70Z/3 cells, butnot for the β-IFN gene. These results indicate the virus-induced complexhas all the in vitro binding properties and in vivo transcriptionalproperties of NF-κB.

Equivalents Those skilled in the art will recognize or be able toascertain, using no more than routine experimentation, many equivalentsof the specific embodiments of the invention described therein. Suchequivalents are intended to be encompassed by the following claims.

We claim:
 1. A method of identifying an agonist or an antagonist of genetranscription involving a transcriptional regulatory factor selectedfrom the group consisting of:a) IgNF-A; b) E factors; c) IgNF-B; and d)NF-κB comprising the steps of: a) providing a host cell containing areporter gene, the transcription of which is dependent upon the activityof said transcriptional regulatory factor; b) contacting the host cellwith an agent to be tested; and c) determining the effect of thepresence or absence of the agent on transcription of the reportergene,wherein a difference in the extent of transcription of the reportergene indicates that the agent is an agonist or an antagonist of genetranscription involving the regulatory factor.
 2. A method foridentifying an antagonist of NF-κB-mediated gene transcriptioncomprising the steps of:a) providing a host cell containing a reportergene, the transcription of which is dependent upon the activity ofNF-κB; b) contacting the host cell with an agent to be tested; c)determining the effect of the presence or absence of the agent ontranscription of the reporter gene,wherein a decrease in the extent oftranscription of the reporter gene indicates that the agent is anantagonist of NF-κB-mediated gene transcription.
 3. A method accordingto claim 2, wherein the reporter gene has been introduced into the hostcell.
 4. A method according to claim 2, wherein the reporter gene islinked to an NF-κB recognition sequence to which it is not linked innature.
 5. A method according to claim 2, wherein the reporter gene islinked to an NF-κB recognition sequence comprising a nucleotide sequenceselected from the group consisting of:a) GGGGACTTTCC; b) AGGGACTTTCC; c)GGGGATTTCC; d) GGGAAATTCC; e) GGGACTTTCC; f) GGGACTTCCC; g) GGGATTTCAC;h) GGGGATTCCT; i) GGGAATCTCC; j) GGGATTCCCC, and a sequence defined bythe consensus sequence: ##STR4##